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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Núñez-Abades, Pedro A.,
John M. Pattillo,
Tracy M. Hodgson, and
William E. Cameron.
Role of Synaptic Inputs in Determining Input Resistance of
Developing Brain Stem Motoneurons.
J. Neurophysiol. 84: 2317-2329, 2000.
The contribution of synaptic input to
input resistance was examined in 208 developing genioglossal
motoneurons in 3 postnatal age groups (5-7 day, 13-16 day, and 18-24
day) using sharp electrode recording in a slice preparation of the rat
brain stem. High magnesium (Mg2+; 6 mM) media
generated significant increases (21-38%) in both the input resistance
(Rn) and the first time constant
(0) that were reversible. A large percent of
the conductance blocked by high Mg2+ was also
sensitive to tetrodotoxin (TTX). Little increase in resistance was
attained by adding blockers of specific amino acid (glutamate, glycine,
and GABA) transmission over that obtained with the high
Mg2+. Comparing across age groups, there was a
significantly larger percent change in
Rn with the addition of high
Mg2+ at postnatal days 13 to
15 (P13-15; 36%) than that found at
P5-6 (21%). Spontaneous postsynaptic potentials were
sensitive to the combined application of glycine receptor antagonist,
strychnine, and the GABAA receptor antagonist,
bicuculline. Application of either 10 µM strychnine or bicuculline
separately produced a reversible increase in both
Rn and
0.
Addition of 10 µM bicuculline to a strychnine perfusate failed to
further increase either Rn or
0. The strychnine/bicuculline-sensitive
component of the total synaptic conductance increased with age so that
this form of neurotransmission constituted the majority (>60%) of the
observed percent decrease in Rn and
0 in the oldest age group. The proportion of
change in
0 relative to
Rn following strychnine or high
magnesium perfusate varied widely from cell to cell and from age to age
without pattern. Based on a model from the literature, this
pattern indicates a nonselective distribution of the blocked synaptic
conductances over the cell body and dendrites. Taken together, the fast
inhibitory synapses (glycine, GABAA) play a
greater role in determining cell excitability in developing brain stem
motoneurons as postnatal development progresses. These findings suggest
that synaptically mediated conductances effect the membrane behavior of
developing motoneurons.
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INTRODUCTION |
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The postnatal development of the
mammalian neuromuscular system is characterized by the maturation of
the motoneuron membrane and the emergence of adult muscle fiber types.
In the adult, there are two general classes of motoneurons, fast and
slow, based on the properties of the motoneuron and the skeletal muscle
fibers that they innervate (Burke and Rudomin 1977). At
birth, there is less of a distinction between the two classes
(Navarrete and Vrbová 1993
). During the subsequent
postnatal development, multiple processes occur in the motoneuron that
tend to separate these classes, including shortening of the duration of
the action potential and its afterhyperpolarization, increase in
rheobase and decrease in input resistance
(Rn) (Cameron et al.
1991b
; Kellerth et al. 1971
;
Núñez-Abades et al. 1993
) and elimination of
the electrotonic coupling among motoneurons (Mazza et al.
1992
; Walton and Navarrete 1991
). These changes
in physiological properties occur concurrently with a reduction in the
complexity of the dendritic tree (Cameron et al. 1991a
;
Núñez-Abades et al. 1994
).
During the postnatal transition, the pattern of spontaneous discharge
of respiratory motoneurons is altered. In the first two postnatal weeks
most, if not all, cat phrenic motoneurons are activated with each
inspiration (Cameron et al. 1991b). Three weeks later,
almost half of the phrenic motoneurons fail to reach threshold and
generate action potentials for an equivalent respiratory drive. While
the pattern of repetitive discharge is shaped, in large part, by the
characteristics of the action potential (Binder et al.
1996
), it is the Rn of the
cell that determines whether a cell will reach threshold and initiate
firing. The increase in number of quiescent cells during this period
coincides with a halving of the mean
Rn (Cameron et al.
1991b
). The reduction of Rn
takes place during a period of no change in the total membrane surface
area (Cameron et al. 1991a
), implying that the decrease occurs as a result of a reduction in the specific membrane resistance of these cells.
The mechanism of the reduction in specific membrane resistance,
critical to motoneuron differentiation, is the focus of the present
studies. The membrane resistance can be altered by addition of new,
tonically active, synaptic inputs converging on motoneurons and/or as a
result of the addition or deletion of other open ionic channels. Both
of these mechanisms will be investigated in this and the companion
paper. In the first paper, the contribution of several forms of
synaptic input to the input resistance of developing motoneurons will
be examined. In the second paper, the role of potassium conductances in
the developmental process will be explored. To manipulate the various
forms of neurotransmission, we have elected to study developing
motoneurons using a slice preparation of the rat brain stem (see
DISCUSSION for the limitations of the technique). In
previous in vitro studies, the rat genioglossal (GG) motoneurons have
been shown to undergo similar developmental changes in electrical
properties (Núñez-Abades et al. 1993) and
morphology (Núñez-Abades and Cameron 1995
;
Núñez-Abades et al. 1994
) to those described
for the cat phrenic motoneurons studied in vivo.
Both excitatory and inhibitory inputs converging on motoneurons undergo
substantial reorganization during embryonic and postnatal development
(Conradi 1976; Gao and Ziskind-Conhaim
1995
; Ziskind-Conhaim 1990
). We have focused on
the role of inhibitory transmission since the longer duration of their
inputs (Gao et al. 1998
) and greater distribution of
inhibitory synapses on or near the soma (Örnung et al.
1998
) suggests a predominant role of these inputs in
establishing the resting conductance and shunting of other synaptic
inputs. In the adult rabbit, the distribution of glycine and GABA
responses on hypoglossal motoneurons has been suggested to be quite
distinct (Altmann et al. 1972
). GABA has relatively stronger actions on the dorsal dendrites, while glycine was more effective in the center of the nucleus, presumably on the cell bodies
and proximal dendrites. However, this proposed segregation of glycine
and GABAA receptors on hypoglossal motoneurons is
contra-intuitive to more recent reports. First, there is strong
anatomical evidence for colocalization of GABAA
and glycine receptors at single postsynaptic densities of spinal
motoneurons (Bohlhalter et al. 1994
; Todd et al.
1996
) and co-localization of GABA and glycine in their presynaptic terminals (Örnung et al. 1994
,
1996
, 1998
; Shupliakov et al.
1993
; Todd et al. 1996
). Second, co-release of
these neurotransmitters from the same presynaptic vesicle has also been
demonstrated using electrophysiological recordings in both spinal
(Jonas et al. 1998
) and hypoglossal motoneurons
(O'Brien and Berger 1999
). Given the different time
courses demonstrated for the two inhibitory currents, the balance
between co-release and synapses releasing only GABA or glycine may be
more important for controlling the time course of the inhibition.
We have selected to study the role of tonic glycine- and GABA-mediated
neurotransmission in determining the resting conductance in developing
GG motoneurons, although the nature of phasic inhibitory input from
respiratory premotor neurons is also undergoing developmental changes
(Funk and Feldman 1995). The effect of the antagonists to the glycine and GABAA receptors will be
compared with the changes generated by the blockade of action potential
evoked and calcium-sensitive neurotransmitter release. A preliminary
report has been published elsewhere (Cameron et al.
1996
).
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METHODS |
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Surgical procedures and solutions
Experiments were carried out on Sprague-Dawley rats (wt: 10-140
g) of either sex divided into three postnatal age groups (5-7 days,
13-16 days, and 18-24 days). Rats were deeply anesthetized with
halothane, tracheotomized, and ventilated with 95%
O2-5% CO2. The brain stem
slice preparation that we employed has been previously described
(Aghajanian and Rasmussen 1989) with modifications (Mazza et al. 1992
). In brief, the animals were
transcardially perfused with a cold (1-4°C) sucrose-artificial
cerebral spinal fluid (ACSF) and then quickly decapitated. The brain
stem was removed and sectioned at 300 µm in the transverse plane
using a Vibraslice (Campden, UK). In a previous study
(Núñez-Abades et al. 1994
), it was
determined that the transverse plane produced optimal conditions for
recording because the viable motoneurons were in the middle of the
slice and have most, if not all, of their dendritic processes intact.
In addition, the elaboration of GG dendrites with postnatal development
occurred predominantly within the mediolateral plane.
All slices were incubated in a holding chamber containing cold sucrose-ACSF for 35-45 min and then transferred to a second holding chamber containing normal-ACSF at room temperature (21 ± 1oC). The slices were transferred one at time into the recording chamber containing normal-ACSF, and all measurements were made at room temperature. To counterbalance the increased CO2 saturation at the lower temperatures, the percentage of CO2 in the gas bubbling the cold sucrose-ACSF was varied between 2 and 5% to maintain the pH between 7.35 and 7.40. At room temperature, the normal ACSF was bubbled with 95% O2-5% CO2 (pH 7.4). Individual slices were transferred to the recording chamber and superfused with normal ACSF. The composition of sucrose-ACSF was as follows (in mM): 240 sucrose, 2 KCl, 1.25 Na2HPO4, 26 NaHCO3, 10 glucose, 2 MgSO4, and 2 CaCl2. The normal-ACSF consisted of (in mM) 126 NaCl, 2 KCl, 1.25 Na2HPO4, 26 NaHCO3, 10 glucose, 2 MgSO4, and 2 CaCl2.
Recordings and selection criteria
Intracellular impalements were made in the ventralmost region of
the hypoglossal motor nucleus, a site demonstrated to contain predominantly motoneurons innervating the genioglossus muscle (Núñez-Abades et al. 1994). The glass
microelectrodes used were filled with a 3:1 mixture of 4 M potassium
acetate and 3 M KCl (resistance: 80-120 M
). Resting membrane
potentials were measured as the difference between the intracellular
and extracellular potentials after withdrawing the recording electrode
from the cell. Neurons were accepted for analysis if they had stable
resting membrane potentials more negative or equal to
55 mV and
action potentials with a positive overshoot, and if they fired
repetitively to sustained depolarization (see
Núñez-Abades et al. 1993
for details).
Single action potentials were evoked by brief suprathreshold depolarizing current pulses (duration: 100 µs) and were averaged for
eight sweeps. Once cells met these criteria, one of several protocols
was performed to block various forms of synaptic transmission. With the
exception of exposure to tetrodotoxin, the effects of the ionic and
pharmacological manipulations were all reversible. The data for any
given cell in response to synaptic blockade were accepted only if the
membrane potential, spike amplitude and
Rn returned to control value (those
obtained when the cell was just impaled) after a wash out period.
Failure of cells to return to control values after the wash out period
could be due to a change in the health of the cell or in the properties
of the electrode and therefore for consistency, the data from these
cells were not used.
After stable intracellular impalement was achieved, the first series of
tests were performed to establish the control values of
Rn and an estimate of the membrane
time constant. Given the nonuniform nature of the membrane resistance
of motoneurons (Campbell and Rose 1997; Clements
and Redman 1989
; Fleshman et al. 1988
; Iansek and Redman 1973
), the first time constant
(
0) will be used as an estimate of the
"average" membrane time constant (
m). First, the membrane potential was measured in response to a series of
six to eight negative current pulses (500 ms, 1 Hz,
0.05-nA increments). The resistance was calculated as the slope of the current-voltage (I-V) plot using the Clampfit program
(pClamp6 software, Axon Instruments). In some cells, there was evidence of inward rectification ("sag") at the larger hyperpolarizing current steps. In these cases, the voltage for the I-V plot
was measured at the peak negative voltage achieved during the current step.
To determine whether the sag current compromised our ability to measure
peak voltage and first time constant, the time course of the voltage
change in response to various levels of hyperpolarization was plotted
on a semilog scale (Burke and Bruggencate 1971). Even for a cell exhibiting a pronounced sag (e.g., Fig.
1), the contribution of the inward
current was minimal at the smallest current step (
0.05 nA, Fig.
1B). The first time constant (
0)
was fitted by a straight line that extended to 35 ms. At the largest
negative current step (
0.3 nA, Fig. 1C), the semilog plots
revealed a linear portion with similar slope to that in Fig.
1B followed by a marked deviation at approximately 35 ms
associated with the inward current. In most instances, the measure of
peak voltage and first time constant fell within this linear portion
especially at the lower current steps.
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Before each series of current injections, the bridge balance was
carefully adjusted to nullify the contribution of the electrode transient. This balance is achieved by viewing a high gain trace after
the DC component of the membrane potential has been subtracted by a
sample-and-hold unit. The contribution of the electrode was easily
discriminated by the reduced noise level. In addition, we have
previously addressed the issue of the accuracy of the bridge balance
and the resulting Rn measurement
(Núñez-Abades et al. 1993). The
Rn derived by bridge balance was <3%
different from the Rn calculated by
spike height technique (Frank and Fuortes 1956
), which
is unaffected by the accuracy of the bridge balance.
To calculate membrane time constant, average (n = 32-64) voltage transient was measured in response to a 0.05-nA
current pulse of 500 ms duration. This level of hyperpolarization did not appear to activate a sag current (Fig. 1B). The time
constant was then calculated using an exponential fitting algorithm of the Clampfit program. In all instances, the best fit was achieved by a
double exponential equation with the slower exponential term being
defined as the first time constant (
0).
The voltage transient of a group of cells exhibiting prominent sag was
also plotted using a semi-log plot, and time constants were calculated
by exponential peel (Zengel et al. 1985
). In general,
the contribution of the sag current was minimal at the current step
used to calculate
0 (Fig. 1B,
0.05 nA). The values of
0 derived by
exponential fitting and peel were within 4% of one another, and these
values varied in a similar fashion with experimental treatment. Because the majority of time constants measured were <15 ms and sag
contamination, when present, was not manifest until 35-50 ms, we
concluded that the sag had not distorted our ability to make an
accurate measure of the first time constant,
0. We concede that there could be complicating
factors associated with our measures of
Rn and first time constant but, in the
final assessment, our analyses are largely based on comparison between
the measurement made during the control period versus the treatment and
wash out for the same cell. Any error introduced by method of
measurement might be expected to be relatively consistent within
different trials on the same cell and, therefore it is not a factor in
the interpretation of the results.
Experimental protocols
The first series of experiments were designed to determine the contribution of various forms of synaptic transmission to the resting conductance of these developing motoneurons. Following control measurements, one of the following substances was added to the normal-ACSF to block one form of synaptic transmission. Tetrodotoxin (TTX, 1 µM) was added to block the synaptic release associated with a propagated sodium action potential. High magnesium (6 mM) in the presence of 1 mM Ca2+ was added to block both evoked and spontaneous calcium-mediated synaptic release. Finally, a cocktail of postsynaptic receptor blockers (antagonists) was added to the high magnesium solution to block all synaptic transmission, including the calcium-independent release. The cocktail contained 20 µM D-2-amino-5-phosphonovaleric acid (APV), 10 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 10-20 µM strychnine, and 10 µM bicuculline to block the N-methyl-D-aspartate (NMDA), non-NMDA, glycine, and GABAA receptors, respectively. The maximum effect of the drug or ionic manipulation occurred within 10-15 min after exposure to the modified perfusate at which time, the action potential amplitude (without TTX), Rn, and time constant were measured. If the membrane potential had shifted due to the synaptic blockade, current was injected into the cell to hold the potential at its control value during the testing of the membrane properties. A healthy cell was always found to return to the control value of the resting membrane potential after wash out of blockers.
A second series of experiments was designed to assess more specifically
the contribution of inhibitory synaptic inputs to resting conductance.
The effects of glycine- and GABAA-mediated synaptic transmission on Rn and
0 were measured individually and in
combination with one another. In the first protocol, the slice was
exposed for 10 min to either 10-20 µM strychnine or 10 µM
bicuculline followed by a 30-min wash out. In subsequent protocols,
this initial exposure was followed by an exposure to a perfusate
combining both strychnine and bicuculline. This latter procedure was
designed to test for possible interactions between the two anionic
channel antagonists.
Statistical analysis
The dependent variable in all statistical tests was either
Rn or 0 with
control values being compared with each of the treatments. Interactions
between age and treatment were determined using a two-way ANOVA. In the
absence of interactions, the differences between treatments at any
given age were tested using a paired t-test. The statistical
levels are indicated in the tables at three levels: P < 0.05, P < 0.01, and P < 0.001. Significant differences in the percent change of
Rn or
0 in
response to a treatment were determined using a one-way ANOVA, and a
Tukey-Kramer Multiple Comparison test was performed to determine
between which groups the significant differences existed.
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RESULTS |
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The response of 208 GG motoneurons, obtained from rats between
postnatal day 5 (P5) and P24, were
included in the present study. Based on our previous study
(Núñez-Abades et al. 1993) that examined the
developmental changes in Rn and time
constant, the neurons reported here were selected from 3 age groups:
P5-7, P13-16, and P18-24. A younger age group
(P0-2) was not included in this study because it had been
shown that there is little difference in their electrical properties
from those examined by the P5-7 group
(Núñez-Abades et al. 1993
). The mean
membrane potential for all protocols was
68.1 ± 1.7 mV.
Effects of general synaptic blockade
The effectiveness of synaptic blockade by high
Mg2+ and TTX was assessed by monitoring the
monosynaptic excitatory postsynaptic potential (EPSP) evoked by
stimulating the adjacent reticular formation dorsal to the nucleus
ambiguus (Fig. 2, top). Both 6 mM Mg2+ and 1 µM TTX blocked the evoked EPSP in
a P13 GG motoneuron. Furthermore, both manipulations
resulted in an increase in Rn with TTX
having less of an effect than 6 mM Mg2+ (Fig. 2,
bottom). These results suggest that a component of
Rn is determined by spontaneous
release of neurotransmitter (Mg2+-sensitive,
TTX-insensitive). Therefore the influence of synaptic inputs in
Rn could be due to action
potential-dependent and/or spontaneous release of neurotransmitter.
This latter form of release could be calcium-dependent and independent.
In the following figures, the influence of each component in
Rn and 0 is
studied.
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Table 1 shows for 64 GG motoneurons that
the addition of high Mg2+ resulted in a
significant increase in both Rn and
0. While the absolute magnitude of the
increase in Rn is similar across ages, the percent change found at P13-16 was significantly
greater (P < 0.01) than the younger age
(P5-7). Figure 3 shows the
effect of exposing the neuron to first TTX, then high magnesium and
finally a cocktail of high magnesium and postsynaptic receptor
blockers. Similar to the effects shown in Fig. 2, TTX-insensitive,
spontaneous release contributes to Rn.
We attempted to dissect the spontaneous release into calcium-sensitive
and calcium-insensitive components. In the two cells (P6 and
P24) shown in Fig. 3, the contributions (absolute and
relative) of these two synaptic components differed slightly from one
another. On the average, the addition of the antagonists failed to
generate any significant increase in
Rn. We conclude from these results
that most of the synaptic influence on
Rn is calcium dependent.
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If proliferation of tonically active synaptic inputs were responsible
for the decrease in Rn found at 2 wk,
then there should be an age-dependent enhancement of the synaptic
components at P13-16. The treatment- and age-dependent
effects of this combined protocol are summarized in Table
2 for 20 cells. There was a significant
effect of TTX treatment on both the mean
Rn and 0 at
all ages. With the addition of high Mg2+ and
antagonists, only one age group (P13-14) showed a further significant increase in both mean Rn
and
0. Although many cells, predominantly in
the two older age groups, did not appear to return to their control
values after a 30-60 min wash out (possibly due to the persistent
effects of TTX), no statistical differences were found between control
and wash out values at any age. Comparing across age groups, there was
a significantly larger (P < 0.05) percent change in
Rn at P13-14 associated
with the addition of TTX, high Mg2+ and
antagonists than that found at P5-6. However, the increase in the mean values at P13-15 was not characteristic of all
the cells in that age group.
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Figure 4 shows the percent change of Rn for each of the 20 cells from this protocol. Several observations emerge from this plot. First, in the majority of cells, the high Mg2+-antagonist combination generated the largest increases in Rn. Second, in most cases, the increase in Rn produced by the TTX response was supplemented by the other two manipulations. Third, two P13-14 cells showed a greater change in Rn with synaptic blockade than was evident at either the younger or older ages. Thus the differences in mean values calculated at P13-14 may be the result of a large synaptic contributions of a select subpopulation of cells (lower resistance) within the nucleus. Given the broad range of Rn in P5-6, no correlation existed between the control Rn and the percent change associated with synaptic blockage.
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Effects of inhibitory neurotransmitter release
There were several instances in which spontaneous synaptic
potentials were observed. One example of such activity in a GG motoneuron from a P20 animal is shown in Fig.
5. These potentials were considerably
reduced in amplitude at 55 mV and were sensitive to a combination of
strychnine and bicuculline. Normally, the chloride equilibrium
potential would be more negative than
55 mV, but, due to the chloride
introduced by the intracellular electrode, the potential was shifted to
a more positive value. In the three cells demonstrating such
spontaneous activity, all potentials were blocked when
strychnine/bicuculline was added to the perfusate. In the following
figures, the voltage traces were averaged and, for this reason, they
did not show the large fast synaptic events prominent in Fig. 5 or in
single voltage records (not shown). Besides averaging, spontaneous
activity might be less evident if the membrane potential were near the
chloride equilibrium potential. The membrane potential was not
systematically varied to look for evidence of spontaneous inhibitory
postsynaptic potentials (IPSPs).
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In Fig. 6, the changes in
Rn are shown with block of fast
inhibitory transmission followed by block of calcium-dependent release for two motoneurons from a P5 and a P23 animal.
Strychnine alone (10 µM) was found to significantly increase both the
mean Rn and 0
of GG motoneurons at all ages (Table 3).
In addition to the treatment-dependent changes, there were also
age-dependent changes. The percent change in
Rn and
0 was
significantly greater (P < 0.05) at P20-24
than that found at younger ages. The disproportionate nature of these
changes in Rn and
0 will be examined in more detail later. For
the two cells presented in Fig. 6, there was no significant change in
either Rn or
0 when bicuculline was added to the strychnine
perfusate. These observations of no additive effect of bicuculline on
Rn or
0 is
further supported by the larger sample of 20 cells presented in Table
4. Bicuculline alone (10 µM)
significantly (P < 0.01) increased both the mean
Rn (42 ± 4 to 47 ± 5 M
)
and
0 (9.1 ± 1.0 to 10.7 ± 1.3 ms)
of 11 GG motoneurons studied from all 3 age groups (not shown). It is
interesting to note in Fig. 6 that there was a larger
Mg2+-sensitive component that exceeded the
strychnine/bicuculline-sensitive component in the cell of the younger
as compared with the older animal. This relationship is more evident in
the pooled data presented in Table 5.
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The nature of the conductances antagonized by strychnine and
bicuculline was examined in more detail for the P23 cell in
Fig. 6. Following a wash out of combined antagonists in which the cell returned to 30 M, exposure to bicuculline alone generated a
Rn of 35 M
. This pattern was seen
in 8 of 10 cells studied with strychnine, bicuculline, and
strychnine-bicuculline combination. In one of the two remaining cells
(P20), the effect of the two antagonists were partially
additive, failing to reach the sum of the two individual effects. The
final cell (P13) actually exhibited a response to the
combined antagonists that exceeded the sum of the individual effects.
The "occlusion" effect characteristic of the eight cells was
observed across all ages studied. If these receptor antagonists are
specific throughout postnatal development, then these data may suggest
some form of interaction between the strychnine- and
bicuculline-sensitive channels in these developing motoneurons.
In Fig. 6, the addition of high Mg2+ blocked a
calcium-dependent component of Rn that
was not mediated by glycine or GABAA receptors. The magnitude of this effect is summarized for 29 cells in Table 5. In
all but one age group, there was a significant increase in
Rn and 0 when
high Mg2+ was added to the bath containing
strychnine. The exception was in the oldest age group
(P20-23), in which there was no significant increase in
either property with the addition of high Mg2+.
Thus a larger part of the calcium-dependent synaptic release is
associated with the strychnine-sensitive component at the oldest age as
compared with earlier periods in development. Figure
7 presents the changes in both
Rn and
0 with
strychnine and high Mg2+ as a function of control
Rn for individual motoneurons in the three different age groups. In this figure, the length of the line
interconnecting the strychnine and high Mg2+
values indicates the magnitude of the strychnine-insensitive synaptic
component. With the exception of one cell, P20-23 has more
cells showing short lines than either younger age group. Thus
neurotransmitters other than the glycine and
GABAA systems play a greater role at
P5-6 and P13-16, while these other
neurotransmitters are of lesser import at P20-23 in
determining Rn.
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It should be noted that there are some differences in the magnitude of changes in response to high magnesium between Tables 1 and 5. Although the exposure to high magnesium followed strychnine in Table 5, it might be expected that the percent change associated with Mg2+ might be of the same order in both experimental protocols. Based on the large variation evident in responses by individual cells (Figs. 4 and 7), a statistically significant difference was found only in the larger sample size, although the same trend of a larger percent increase at P13-16 was present in both protocols. As noted earlier, the mean values derived can be influenced by the large changes in a subpopulation of the motoneurons.
According to model developed by Redman and colleagues (Redman et
al. 1987), neurons that show a disproportionately large
increase in membrane time constant as compared with
Rn are indicative of conductances
located on the more distal dendritic compartment. Figure
8 shows two plots of percent change in
0 as a function of the percent change in
Rn after the motoneurons were exposed to high Mg2+ (A) or strychnine
(B). A line with slope of one separates the data points into
cells that show a larger change in
0 (above line) from ones that show a larger change in
Rn (below line). For both
manipulations (high Mg2+ and
strychnine-sensitive), there were roughly equal numbers of points lying
above as below the line for all age groups. It is interesting to note
how large the variability is when comparing the relative change in
Rn and time constant between cells
within an age group. This distribution of points implies that the
blocked calcium-dependent synaptic conductances were not preferentially localized to either the cell body or dendrites for this population of
brain stem motoneurons. At all ages, there was a wide diversity in the
magnitude of the response to either blocker. In particular, the
greatest variation was found for the oldest age group in response to
strychnine.
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![]() |
DISCUSSION |
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This study demonstrates the role that tonically active synapses
play in determining the Rn and
0 of developing motoneurons in an in vitro
preparation. This resistance was mediated by both action
potential-evoked (TTX-sensitive) and spontaneous (TTX-insensitive) neurotransmitter release. The relative contribution of these two forms
to Rn and
0
varied from cell to cell with a significant increase occurring between
P5-6 and P13-16. Almost all of the spontaneous
release was calcium dependent (Mg2+ sensitive)
with few cells exhibiting a calcium-independent component (blocked by
the addition of receptor antagonists). When large spontaneous
postsynaptic potentials were recorded, these events had an apparent
reversal potential appropriate for chloride and were blocked by
strychnine and bicuculline. The greatest percent change in
Rn and
0
after blockade with strychnine occurred between P13-16 and
P20-24. In the oldest age group, high magnesium failed to
significantly increase Rn and
0 beyond that resulting from exposure to
strychnine. The percent changes in
0 relative
to Rn suggested that both the
strychnine- and magnesium-sensitive conductances were distributed
equally over the cell body and distal dendrites. In general, synaptic
transmission makes a significant contribution (up to 36% change after
blockade) to Rn of developing motoneurons with glycine/GABA neurotransmission becoming the major (exceeding 60%) synaptic component in the adolescent rat.
Measurement of membrane properties
Both Rn and
0 were derived from the membrane responses to
long duration (500 ms) hyperpolarizing current steps. In many cells, it
was apparent that there was an inward or sag current activated at the
larger current pulses. The
0 was derived from
a small hyperpolarizing current step (
0.05 nA) that failed to
activate an inward current as determined by a semilog plot of voltage
as a function of time. Even at the largest current steps, the time point when peak voltage for calculation of
Rn occurred was measured prior to the
onset of the sag. It is still possible that the measurement of these
membrane properties is in some way contaminated by the inward current.
In which case, these properties should be referred to as apparent
Rn and
0.
Likewise, our manipulations of synaptic activity could also be
modulating the magnitude of the sag current. One means to eliminate the
contribution of sag is to linearize the membrane properties by the
introduction of cesium to the external bath (Rapp et al.
1994
). As will become evident in the accompanying paper,
external cesium does not always eliminate the inward current and
clearly has an effect on other currents influencing the action potential and potentially resting conductances.
The interpretation of our results might be complicated by artifacts
produced by the use of the sharp electrode and the in vitro slice
preparation. We have attempted to minimize the inaccuracies associated
with the bridge balance (high gain sample-and-hold trace), but it might
be suggested that the resistance measurements would be more accurate if
we conducted the experiments using whole cell configuration featuring a
lower electrode resistance. However, whole cell recording in these
neurons is complicated by a nonuniform change in resistance associated
with cellular dialysis (Robinson and Cameron 1998,
2000
). The cellular dialysis associated with whole cell
configuration can be avoided by using the perforated patch technique.
In our hands, the resistance of a perforated patch recording approached
the values of our sharp electrodes creating similar problems for bridge
balance (unpublished observation). Each recording method had its own
drawbacks, and we decided that sharp electrodes were the better choice
given the other constraints of our experimental design.
A second potential complication to the interpretation of our data is
introduced by the preparation of the brain stem slice. If there is an
age-dependent change in the numbers of premotor neurons surviving the
slice preparation due to the actual sectioning procedure or the
tolerance of neurons to anoxia, then the true role of synaptic input
may be inaccurately assessed. Slices from older animals should have
fewer premotor sources of input. As a result, the magnitude of the
increase role found for synaptic input in determining
Rn in the mature animals would be
underestimated. This conclusion would also be supported by the lack of
spontaneous synaptic activity in most cells studied. There is evidence
that transverse slices can have sufficient circuitry intact to generate spontaneous rhythmic, respiratory motor output from hypoglossal motoneurons (Smith et al. 1991). However, these
oscillating slices were prepared from younger animals
(P0-3) and were thicker slices (range: 500-650 µm) than
those in the present study. There is no evidence for phasic input from
the respiratory generator in our slices. Clearly, there has been a
substantial reduction in the number of premotor neurons projecting to
GG motoneurons in the slice especially at the older ages. Thus we
conclude that the increased role observed for inhibitory synaptic input
in the older animals studied in vitro underestimates the magnitude
present in the intact nervous system.
Development of membrane properties
During rat embryonic development, the mean
Rn of lumbar spinal motoneurons
decreased from 271 M at embryonic day 14-15 to 65 M
at birth (Ziskind-Conhaim 1988
). This decrease is
accompanied by a fivefold increase in the mean input capacitance of
these cells indicating a substantial increase in cell size. The
Rn continues to decrease into the
postnatal period from a mean of 18 M
at 3-5 days to 5 M
at
P9-11 (Fulton and Walton 1986
). A similar postnatal decrease has been noted for cervical motoneurons in the cat
(Cameron et al. 1991b
). Unlike the earlier reports in the rat spinal cord, the decreased Rn
in the cat was found to occur during a period of no net increase in the
size of the phrenic motoneurons (Cameron et al. 1991a
).
A similar pattern to the cat phrenic motoneurons has been described for
Rn and membrane surface area of the
rat brain stem motoneurons analyzed in the present study
(Núñez-Abades and Cameron 1995
,
1997
; Núñez-Abades et al.
1993
, 1994
). Assuming that specific capacitance
remains constant over development, these latter studies imply that
there was a change in the specific membrane resistance of the cell to
achieve the decreased Rn without
altering total membrane surface area.
Based on the present data set, there is a synaptically mediated
component to the resting conductance of GG motoneurons. The largest
component of the synaptically mediated conductance is derived from
action potential-evoked neurotransmitter release and the largest
increase of this evoked release occurring between P5-6 and
P13-16. This corresponds to the time at which the membrane shows a dramatic change in specific membrane resistance
(Núñez-Abades et al. 1993). Thus at least
part of this decreased Rn at 2 wk can
be accounted for by an increase in the synaptic conductances.
Glycine and GABA transmission
For rat lumbar motoneurons, both GABAA- and
glycine-mediated transmission are present at embryonic day
18 with the GABA conductance accounting for seven times the
conductance of glycine (Gao and Ziskind-Conhaim 1995).
This relative imbalance between GABAergic and glycinergic transmission
characteristic of embryonic development is lost by birth when both
conductances become roughly equal. In embryonic and early postnatal
life, activation of either GABAA or glycine
receptors on spinal motoneurons generated an inward current and
membrane depolarization (Gao and Ziskind-Conhaim 1995
). Even though these receptors mediate a slight depolarization, they achieve an inhibition by shunting incoming synaptic currents. These
depolarizing currents are also believed to be critical for the
stabilization of newly formed synapses. Eventually, the adult chloride
reversal potential is achieved by the action of a more efficient Na-Cl
pump (Rohrbough and Spitzer 1996
). In the present study,
most cells showed no change in membrane potential with the blockade of
glycine and/or GABAA transmission. Those cells that did show a change were found to depolarize slightly, probably due
to the leakage of chloride from the intracellular electrode as was
evident in Fig. 5.
The glycine receptor changes during development with respect to its
subunit composition. The adult glycine receptor that is blocked by
strychnine does not appear until 7 days in culture of embryonic rat
spinal cords (St. John and Stephens 1993). There is a
transition from strychnine-insensitive, fetal glycine receptors containing the alpha-2* subunit to strychnine-sensitive adult receptors
containing the alpha-1 subunit after about 1 wk in culture (Withers and St John 1997
). This conversion between
fetal and adult forms of the receptor is also associated with more
rapid kinetics of the channels (Takahashi et al. 1992
).
The increase in strychnine-sensitive conductances that we found with
age could be explained by the gradual disappearance of the fetal
(strychnine-insensitive) form of the receptor. We do not believe this
to be the case because the neurons in culture derived from
embryonic day 14 spinal cord reach their adult form after
only 1 wk in culture. This stage would roughly equate to the end of an
average gestational period of a rat (21 days). The first age group in
which we studied the effect of strychnine was at P5-6 and
should represent principally adult receptors.
Developmentally, the GABAergic system undergoes substantial regulation
(Ma et al. 1992, 1993
; Poulter et
al. 1992
, 1993
; Rekling et al.
2000
; Schousboe and Redburn 1995
; Xia and
Haddad 1992
). Transcripts for the GABAA
receptor subunits alpha 2, 3, 5, beta 2-3 and gamma 2-3 are all
present in presumptive motoneurons of the mantle zone by E13
in rat. Peak expression of subunits occurs between E17 and
E20 when mRNA for alpha 2-5, beta 1-3, and gamma 1-3
subunits are all present. Alpha 3-5, beta 1-2, and gamma 1 and 3 subunits then decrease and are almost absent by the end of the second
postnatal week. Similar data on the developmental changes in subunit
composition for hypoglossal motoneurons are not available. It is
unlikely to have a similar sequence because of the differences in the
GABA receptor subunits between spinal and cranial motoneurons. The
1 and
3 subunits are
strongly expressed in cranial motoneurons (Hironaka et al.
1990
), while they are very sparse or absent in spinal
motoneurons where the
2 subunit dominates
(Ma et al. 1993
; Persohn et al. 1992
).
Developmental forms of the receptors may complicate the specificity of
the antagonists. In the hippocampus, there is evidence for a transient
expression of a bicuculline-insensitive GABA response mediated via
chloride channels that disappears by postnatal week 2 (Martina et al. 1995
). As was the case with a potential
strychnine-insensitive component, our results may underestimate the
GABAA contribution to resting conductance in the
first postnatal week if there were a significant bicuculline-insensitive component.
Tonic inhibitory inputs (i.e., glycine) have been implicated in
establishing motoneuron excitability during some experimental induced
sleep states. In cat lumbosacral (Morales et al. 1987) and masseter motoneurons (Kohlmeier et al. 1997
),
carbachol-induced atonia is produced by strychnine-sensitive and
bicuculline-insensitive mechanisms. This same lab has demonstrated a
similar strychnine-sensitive inhibition for hypoglossal motoneurons
(Yamuy et al. 1999
). Alternatively, it has also been
proposed that removal of dysfacilitation mediate this atonia response
in cat hypoglossal motoneurons (Kubin et al. 1994
,
1996
). In our in vitro preparation, some of these
inhibitory premotor inputs to the GG motoneurons studied remain intact
in the brain stem slices as reflected by
strychnine/bicuculline-sensitive spontaneous activity. With a few
exceptions (Umemiya and Berger 1995
), most of the
sources of inhibitory connections remain unknown; however, it is
evident that the role of glycine/GABA in determining GG motoneuron
excitability increases with age. It will be important that correlative
anatomical studies be undertaken to map the changes in density and
distribution of the inhibitory receptor during postnatal development.
Interactions between the glycine- and GABAA-mediated responses
There is strong anatomical evidence for the colocalization of
GABAA and glycine receptors at single
postsynaptic densities (Bohlhalter et al. 1994;
Todd et al. 1996
) and for GABA and glycine colocalization in presynaptic terminals (Örnung et al.
1994
, 1996
, 1998
;
Shupliakov et al. 1993
; Todd et al.
1996
). Electrophysiological measurements indicate that GABA and
glycine can be co-released from the same presynaptic vesicle onto
spinal (Jonas et al. 1998
) and hypoglossal motoneurons
(O'Brien and Berger 1999
). Although the postsynaptic
compliment of receptors varies between synapses with some being GABA
only, some glycine only and some mixed (Todd et al.
1996
), the activation of one receptor species over another may
be more important in determining the time course of the response. In
hypoglossal (O'Brien and Berger 1999
) as is the case
with spinal motoneurons (Jonas et al. 1998
), the time
constant for decay of the GABAA
receptor-mediated currents was slower than that of glycine receptor-mediated currents. Which neurotransmitter initiates the inhibition may be less important than the role that the relative amount
of GABA versus glycine play in establishing the time course of the
inhibitory currents and in shaping of the resulting motor coordination.
Our results suggest some form of interaction between glycine and
GABAA receptors. The response to strychnine and
bicuculline combined was no different from the response to either
blocker individually. Occlusion between these two receptors has been
reported previously. The GABA response is partially occluded by glycine in cultured rat medullary neurons (Lewis and Faber
1993). It might be proposed that there is some kind of steric
hindrance of the GABAA receptor when strychnine
binds to an adjacent glycine receptor. Alternatively, the concentration
of strychnine used in the present study (10 µm) to block glycine
receptors can also significantly block the GABAA
receptors in motoneurons in the first postnatal week (Jonas et
al. 1998
; O'Brien and Berger 1999
). The
converse for bicuculline is not true. If this nonselective blockade of GABAA receptors persists at the older ages, then
the occlusion observed between the two receptors in the present study
may be artifactual. Irrespective of the possibility of occlusion, it is
interesting to note that the magnitude of the change in resistance associated with glycine receptor blockade was similar to that associated with GABAA receptor blockade. This
would be the case if the majority of inhibitory synapses co-released
both neurotransmitters or the total conductance mediated by the two
neurotransmitters was comparable as found by O'Brien and Berger
(1999)
.
Differential distribution of synapses
The present data demonstrate that synaptic conductances contribute
to the resting membrane conductance (input resistance). In particular,
the contribution of glycine/GABA increases, reaching its largest value
at the oldest age group. During this period, the increased contribution
of inhibitory synapses is associated with a decreased contribution of
excitatory synapses. However, the role of the two fast inhibitory
neurotransmitters on motoneurons has been described to be quite
different. An earlier study had segregated the influence of glycine and
GABA to different sites on the dendritic tree of rabbit hypoglossal
motoneurons (Altmann et al. 1972). GABA has relatively
stronger actions on the dorsal dendrites while glycine was more
effective in the center of the nucleus, presumably the cell bodies and
proximal dendrites. We have applied an analysis developed by Redman and
colleagues for sympathetic ganglion cells (Redman et al.
1987
) to assess the relative distribution of a blocked
conductance. They observed that all sympathetic ganglion cells
exhibited a disproportionately larger change in
0 as compared with
Rn when exposed to extracellular barium. Their interpretation was that the barium-sensitive conductances are preferentially distributed to the distal dendrites. When a similar
plot is generated for GG motoneurons exposed to high
Mg2+ and strychnine, the points lay both above
and below the line of unity slope. We interpreted this distribution to
mean that these synaptic conductances were distributed more randomly
across the cell membrane for the population of GG motoneurons. For some motoneurons, most of the conductance is associated with the soma or
proximal dendrites, whereas, for others, most of the synaptically mediated conductance is associated with more distal dendrites. Therefore the possible impact of tonic inhibitory synapses determining excitability in GG motoneurons must be quite diverse. In some cells,
these inputs may have a predominant role in establishing resting
conductance and shunting synaptic inputs, but not in others.
A recent report on rat Purkinje cells studied in the cerebellar slice
preparation (Hausser and Clark 1998) demonstrated a relatively uniform distribution of inhibitory inputs. Simultaneous whole cell recording from cell body and dendrite revealed a similar change in the both Rn and
m when 30 µM bicuculline was added to the
perfusate. These authors also emphasized how this tonic inhibition
could play a role in dynamically modulating synaptic integration with
synaptic inputs being attenuated in the presence of high synaptic
background. Additional support for our observation of an increased role
for glycinergic inputs has come from the analysis of miniature
inhibitory postsynaptic currents (IPSCs). The IPSCs became faster and
larger between neonate and juvenile. The shorter time course was
correlated to a faster decay time course and coincided with an
increased expression of adult (alpha 1) glycine receptor subunit of the
channels (Singer et al. 1998
). However, this postnatal
change in receptor expression did not alter the conductance of the
channels (Singer and Berger 1999
). Instead, these
authors proposed that the increase in IPSC amplitude was due to an
increase in the number of receptors per synapse with postnatal
development. To support these observations, one could generate an
anatomical map of glycine and GABAA receptors on
the surface of motoneurons at various stages of postnatal development. In particular, it would be interesting to see whether these changes correspond with the formation of receptor clusters on more distal dendrites as described on cat spinal motoneurons (Alvarez et al. 1997
).
In summary, there was a significant contribution of synaptic input to the Rn of these developing motoneurons. Most of this conductance is mediated by synaptic release associated with an action potential while the balance is associated with calcium-dependent spontaneous release. The role of glycinergic/GABAergic transmission increases over the course of postnatal development to account for most of the synaptic conductance in the mature animals (3 wk). These data support the general concept that development of motor skills is characterized by increased inhibition of reflex pathways.
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ACKNOWLEDGMENTS |
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The authors thank Dr. David W. Robinson and the reviewers for invaluable 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 27 July 1999; accepted in final form 28 July 2000.
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REFERENCES |
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