EPSP Amplitude Modulation at the Rat Ia-Alpha Motoneuron
Synapse: Effects of GABAB Receptor Agonists and Antagonists
KAVITA R. PESHORI, WILLIAM F. COLLINS III, AND LORNE M. MENDELL
Department of Neurobiology and Behavior, State University of New York at Stony Brook, New York 11794-5230
Peshori, Kavita R., William F. Collins III, and Lorne M. Mendell. EPSP amplitude modulation at the rat Ia-alpha motoneuron
synapse: effects of GABAB receptor agonists and antagonists. J.
Neurophysiol. 79: 181–189, 1998. The object of this study was to
examine the relationship between excitatory postsynaptic potential
(EPSP) amplitude, posttetanic potentiation, and EPSP amplitude
modulation at synapses made by group Ia afferents on motoneurons
in the rat. These relationships were evaluated in cells in untreated
rats and in cells in rats treated with the g-aminobutyric acid-B
(GABAB ) receptor agonist baclofen and antagonist CGP-35348,
which were used to manipulate Ca 2/ entry into presynaptic terminals and consequently probability of transmitter release from them.
There was no evidence for postsynaptic action of these drugs from
measurement of their effects on motoneuron properties. During
high-frequency stimulation (32 shock bursts at 167 Hz), EPSP
amplitude either decreased (negative modulation) or increased
(positive modulation) in response to successive stimuli at different
connections. In untreated rats this frequency-dependent amplitude
modulation behavior was inversely but weakly correlated with
EPSP amplitude measured at low frequency. Intravenous (iv) administration of the GABAB agonist, baclofen, produced a marked
and progressive decrease in EPSP amplitude measured at low frequency coincident with a change in frequency-dependent EPSP
amplitude modulation toward more positive values (synaptic facilitation). In contrast, an increase in EPSP amplitude occurred after
iv administration of the GABAB antagonist CGP-35348 that was
accompanied by a negative shift in EPSP amplitude modulation
during high-frequency stimulation. The negative shift in EPSP amplitude modulation (synaptic depression) after CGP-35348 application was much smaller than the positive shift induced by baclofen
when normalized to the change in EPSP amplitude. Posttetanic
potentiation decreased after baclofen but did not increase after
CGP-35348. The relationship between modulation and EPSP amplitude was much steeper after GABAB receptor manipulation in
either direction than that observed in the population of motoneurons in untreated preparations. This suggests that in the rat differences in probability of release play at most a small role in determining EPSP amplitude across the motoneuron pool.
INTRODUCTION
A series of studies in the cat has illustrated that the successive excitatory postsynaptic potential (EPSPs) generated in
motoneurons of different types during high-frequency stimulation of Ia afferent fibers differ in their amplitude modulation (Collins et al. 1984; Honig et al. 1983; Mendell et al.
1995). Large-amplitude EPSPs generated in motoneurons
with a small value of rheobase, large input resistance, and
afterhyperpolarization (AHP) of long duration tend to undergo a progressive decrease in amplitude during high-frequency stimulation (negative modulation, see METHODS ).
Conversely, small-amplitude EPSPs in motoneurons with a
low input resistance, high rheobase and short-duration AHP
increase in amplitude during high-frequency stimulation
(positive modulation). Positive correlations have also been
observed between posttetanic potentiation and EPSP amplitude after high-frequency stimulation (Davis et al. 1985,
1987). These observations suggest that the processes that
underlie EPSP amplitude modulation and potentiation contribute to the distribution of Ia EPSP amplitudes across motoneurons (Mendell et al. 1990).
By analogy with peripheral systems (Del Castillo and
Katz 1954; Frank 1973; Gertler and Robbins 1978; Kuno
1964), it was hypothesized that the differences in frequencydependent behavior at these synapses resulted from differences in presynaptic properties, specifically in probability of
transmitter release (Collins et al. 1984). A buildup of Ca 2/
in the presynaptic terminals from repetitive stimulation (residual Ca 2/ ) is considered to lead to an increase in the
probability of transmitter release resulting in facilitation of
the postsynaptic response during a high-frequency burst
(Katz and Miledi 1968; Mallart and Martin 1967, 1968).
At connections with high initial probability of transmitter
release (large initial values of P) and consequently large
EPSP amplitudes, depression is seen during high-frequency
stimulation, despite the residual Ca 2/ buildup, possibly because of the unavailability or depletion of adequate releasable transmitter (Betz 1970) or inability to release available
transmitter on subsequent stimuli (Korn et al. 1981). At
connections with low initial probability of release, EPSP
amplitude increases during high-frequency stimulation. Thus
in this work we have assumed that the behavior of EPSP
amplitude during high-frequency stimulation is an index of
the average probability of release at that connection.
The focus of the present study was to investigate the dependence of EPSP amplitude during high-frequency stimulation on probability of transmitter release more explicitly.
The approach was to manipulate probability of release pharmacologically by using agonists and antagonists of the gaminobutyric acid (GABAB ) receptor. This receptor, which
is known to be present on terminals of Ia fibers (Bowery et
al. 1987; Price et al. 1984), acts to control entry of Ca 2/
into sensory neurons (Deisz and Lux 1985; Dolphin and
Scott 1987; Dunlap and Fischbach 1981). Previous studies
suggested that the GABAB agonist baclofen reduces monosynaptic EPSP amplitude (Edwards et al. 1989; Lev Tov et
al. 1988; Peng and Frank 1989) by decreasing probability
of transmitter release (Edwards et al. 1989; Peng and Frank
1989) because of reduction in Ca 2/ entry into the presynaptic terminals (Curtis et al. 1997). The GABAB antagonist
CGP-35348 is also known to reduce the level of presynaptic
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K. R. PESHORI, W. F. COLLINS, AND L. M. MENDELL
inhibition in the spinal cord (Curtis 1998; Curtis and Lacey
1994). By using these agents, the aim in the present experiments was to examine in detail the relationship between
EPSP amplitude, EPSP amplitude modulation, and posttetanic potentiation in individual cells. One question was
whether activation and blockade of the GABAB receptor
yields the same functional relationship between EPSP amplitude modulation and amplitude. A second question was
whether or not this relationship is similar to that observed
across the population of connections. This would speak to
the question of whether or not differences in EPSP amplitude
in different motoneurons can be attributed to differences in
probability of transmitter release. Preliminary results from
these studies have been published in abstract form (Mendell
et al. 1991; Peshori et al. 1991, 1993).
METHODS
Adult, male Sprague-Dawley rats (300–500 g) were anesthetized with a mixture of ketamine and xylazine (90 and 10 mg/kg,
im, respectively) and anesthesia was maintained by a continuous
infusion (0.4–1.6 ml/hr) of this mixture diluted in 10% dextrose
saline. The rats were mechanically ventilated and heart rate, ECG,
blood pressure, expired CO2 , and rectal temperature were monitored. Body temperature was maintained at 377C by using a heat
lamp and a heating pad. The spinal column was placed in a custommade spinal clamp and a dorsal laminectomy was performed to
expose segments T 12 –S2 of the spinal cord. The left medial gastrocnemius (MG) and lateral gastrocnemius-soleus (LGS) nerves were
exposed and cut. The proximal cut ends were placed on bipolar
platinum stimulating hook electrodes and covered with warm mineral oil. The dura overlying the spinal cord was removed and the
cord was covered with mineral oil. A platinum ball electrode was
placed on the dorsal columns and the stimulus intensities for the
MG and LGS nerves were set at 2 times threshold for the dorsal
column response to ensure maximal activation of group Ia afferent
fibers. Incoming afferent volleys were monitored in this manner
to ensure faithful conduction of afferent volleys during high-frequency stimulation.
Intracellular recordings were made by using glass microelectrodes filled with 3 M potassium acetate with tip resistance between
4 and 16 MV. Impaled motoneurons were identified as MG or LGS
by their response to antidromic stimulation of the MG or LGS
muscle nerve and the latency was used to calculate axonal conduction velocity. The analysis was restricted to motoneurons with
antidromic action potential amplitudes ú75 mV. Motoneuron rheobase was measured as the minimum intracellular depolarizing current (50 ms duration) required to generate an action potential.
Input resistance (Rn ) was measured as the averaged (n Å 64)
amplitude of the voltage change in response to 1 nA of hyperpolarizing current (50-ms duration) injected intracellularly. Before measuring Rn , the averaged record was corrected for membrane potential Sag by using a procedure similar to that used by Zengel et al.
(1985).
Composite Ia-EPSPs were elicited by stimulation of the heteronymous muscle nerve. Two stimulus paradigms were employed;
first, low-frequency (18 Hz) trains and second, 32 shock bursts at
high-frequency (167 Hz) delivered every 2 s. Successive triggered
events were averaged in register (n Å 256 for 18 Hz trains, n Å
64 for 167-Hz bursts) and the amplitudes of the averaged EPSPs
were measured (EPSP18 Hz ; and for the burst, EPSP1 , EPSP2 , . . . ,
EPSP32 ). A correction factor was added to the measurements of
all but the first EPSP within a burst to compensate for the summation of each EPSP with the falling phase of the previous EPSP.
The value of this correction factor was calculated as the magnitude
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of the voltage decay of the last (32nd) EPSP in the burst between
times representing where the onset and peak of an imaginary 33rd
EPSP would have occurred.
These two stimulus paradigms have been used in previous studies in cats (Collins et al. 1984, 1988; Davis et al. 1985, 1987;
Koerber and Mendell 1991; Mendell et al. 1995) for several reasons. First, both frequencies are within the normal physiological
range for Ia afferents (Prochazka et al. 1977; Loeb and Duysens
1979). Second, no systematic variability is observed in the amplitude of Ia-motoneuron EPSPs during 18 Hz stimulation (Collins
et al. 1984). Thus EPSP18 Hz is considered to be the control EPSP
for comparison with EPSPs generated at higher frequencies of
stimulation. Third, 32 shock bursts at 167 Hz is an optimal paradigm for revealing the heterogeneity in the response of Ia-motoneuron connections to repetitive stimulation. At frequencies ú167
Hz, most Ia-motoneuron synapses tend to exhibit depression and
conduction failure in afferent terminal branches can occur (Collins
et al. 1984). In preliminary studies we found these criteria to be
valid for the rat as well, and for the sake of consistency and easier
comparison between species, we chose the same stimulus paradigms as those used in the cat.
To assess the change in EPSP amplitude during high-frequency
stimulation, EPSP amplitude modulation, potentiation and facilitation/depression (F-D) were calculated by using the following formulae:
Modulation Å [(EPSP30 / EPSP31 )/2]/EPSP1
(1)
Potentiation Å EPSP1 /EPSP18 Hz
(2)
F-D Å [(EPSP30 / EPSP31 )/2]/EPSP18 Hz
(3)
Modulation is defined as the change in EPSP amplitude during a
high-frequency, 32 shock burst and was calculated as the ratio of
the amplitude of the EPSPs at the end of the burst (average of
30th and 31st EPSPs) to the amplitude of the first EPSP within
the burst (Eq. 1). A modulation value õ1 represents a decrease
in EPSP amplitude during the burst and is termed negative modulation. A modulation value ú1 signifies positive modulation, i.e., an
increase in EPSP amplitude during the burst. Because the interburst
interval used in averaging bursts was 2 s, the initial EPSP in each
burst (except the first burst) was often potentiated in amplitude
because of the influence of the previous burst (Collins et al. 1984;
Davis et al. 1985). This potentiation was quantified by calculating
the ratio of the amplitude of the first averaged EPSP in a highfrequency burst to the amplitude of the averaged control EPSP18 Hz
(Eq. 2). The F-D ratio represents the amplitude of the EPSP at
the end of the burst referred to the value measured at low-frequency
stimulation (EPSP18 Hz ). Because the first EPSP in the burst
(EPSP1 ) was generally potentiated, EPSP1 ú EPSP18 Hz [potentiation (Eq. 2) ú 0] and so F-D was generally more positive than
modulation (see Fig. 3).
Pharmacological studies
After obtaining measurements of electrical and synaptic properties at a given Ia-motoneuron connection, either l-( 0 ) baclofen
(10 mg/kg in saline, Ciba-Geigy, Suffern, NY), ( {) baclofen (10
mg/kg in phosphate buffered saline, Sigma Chemical, St. Louis,
MO or Research Biochemicals International, Natick, MA), CGP35348 (30–400 mg/kg in saline, Ciba-Geigy, Basel), or saline
was injected intravenously while the intracellular penetration was
maintained. A single injection was usually sufficient for baclofen
effects which were not reversible over the intervals studied here
(up to 4 h). In the case of CGP-35348 the initial small dose
often did not have any effect or it wore off very quickly and
supplementary injections were given. This accounts for the wide
range of dosages for CGP-35348. The measurements were repeated
immediately after drug administration and periodically throughout
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FIG . 1. Examples of averaged, composite, heteronymous excitatory postsynaptic potential (EPSPs) recorded from a medial
gastrocnemius (MG) motoneuron in response to stimulation of
lateral gastrocnemius-soleus (LGS) muscle nerve at (A) lowfrequency (18 Hz train; n Å 256 sweeps) and (B) high-frequency
(32 shock bursts at 167 Hz; 2 s interburst interval, n Å 64
sweeps).
the duration of a stable penetration (up to 4 h). All pre- and
postdrug comparisons were made with the paired-sample t-test.
RESULTS
Composite Ia-motoneuron EPSP amplitude
The amplitudes of composite EPSPs were measured in a
total of 103 motoneurons in response to 18 Hz stimulation
(Fig. 1A) and varied from 0.15 mV to 3.35 mV with a mean
of 1.15 { 0.68 (SD) mV. Motoneuron rheobase varied from
0.8 to 19.5 nA (mean Å 8.1 { 3.9 nA; n Å 100) and
motoneuron input resistance varied from 1.2 to 4.1 MV
(mean Å 2.2 { 0.6 MV; n Å 38). EPSP amplitude at 18
Hz was weakly but significantly correlated with both motoneuron rheobase (linear regression, r Å 00.23, P õ 0.02;
Fig. 2A) and motoneuron input resistance (r Å 0.43, P õ
0.01; Fig. 2B).
potentiation and EPSP amplitude was observed (Davis et al.
1985). In contrast, a significant negative correlation was
detected between F-D and EPSP amplitude (r Å 00.25;
P õ 0.01; Fig. 3C), which was nearly identical to that observed for modulation. Thus the dependence of modulation
on EPSP amplitude at rat Ia-motoneuron synapses results
from changes occurring during bursts of high-frequency
stimulation (i.e., facilitation and depression), rather than
changes between bursts (i.e., potentiation).
Manipulation of EPSP amplitude with baclofen and
CGP-35348
A major aim of these experiments was to determine explicitly the effects of manipulating the probability of trans-
Frequency dependence of EPSP amplitude
EPSP amplitude modulation during 32 shock bursts (Fig.
1B) was calculated using Eq. 1 and varied from 0.46 (negative modulation) to 1.56 (positive modulation) with a mean
of 1.25 { 0.2 (n Å 103). As in the cat, large EPSPs tended to
exhibit more negative modulation than small EPSPs. Plotting
modulation against EPSP amplitude (Fig. 3A) revealed a
significant negative correlation, but with a relatively small
correlation coefficient (r Å 00.25, P õ 0.01). The slope of
the linear regression is 00.08, suggesting a small dependence of amplitude modulation on EPSP amplitude. No correlation was detected between amplitude modulation and
either motoneuron rheobase or motoneuron input resistance.
One of the complications in evaluating differences in amplitude modulation (Eq. 1) across the motoneuron pool is
that the first EPSP in the burst is often potentiated with
respect to the low frequency (18 Hz) EPSP amplitude (Davis
et al. 1985, 1987). This is evident in the averaged records
of the present study (Fig. 1) and occurs as a consequence
of the high-frequency activity of the preceding burst (2-s
interburst interval). Thus differences in EPSP amplitude
modulation across the population of motoneurons could result from systematic differences either in potentiation of the
first EPSP in the burst or in faciliation-depression (F-D)
behavior during the burst, or both. The contribution of these
factors to the dependence of amplitude modulation on EPSP
amplitude was examined in more detail by evaluating the
correlation of potentiation (Eq. 2) and F-D (Eq. 3) with
EPSP amplitude. Potentiation of the first EPSP in the burst
was found not to depend on EPSP amplitude(P ú 0.78; Fig.
3B), unlike in the cat where a positive correlation between
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FIG . 2. Plots of relationships between (A) EPSP18 Hz amplitude and motoneuron rheobase and (B) EPSP18 Hz amplitude and motoneuron input resistance in untreated rats. Each point represents data obtained from a single
motoneuron. Linear regressions are plotted as solid lines through data. r Å
correlation coefficient for regression.
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K. R. PESHORI, W. F. COLLINS, AND L. M. MENDELL
changes in EPSP amplitude, modulation, and potentiation of
070 { 14%, 67 { 46%, and 023 { 14%, respectively,
although these are approximate values because they were
obtained over different treatment intervals (i.e., until the
motoneuron penetration was lost). The decreases in EPSP
amplitude and potentiation and the positive shift in modulation could be detected within 2 min after application of
baclofen (Fig. 6A) and persisted for as long as the motoneuron penetration was maintained (up to 2.8 h). These effects
did not wear off over the several hours that the intracellular
penetration was maintained.
Administration of the GABAB antagonist CGP-35348 permitted evaluation of these same relationships when probability
of release was increased (Fig. 4, C and D). Intravenous administration of CGP-35348 produced a rapid and marked increase
in EPSP amplitude (50 { 18%, n Å 8). However only a small
change in amplitude modulation was observed ( 013 { 9%,
n Å 8; Figs. 4–6), even after repeated doses (see METHODS )
unlike the finding after baclofen administration where modulation underwent substantial changes in conjunction with those
in EPSP amplitude (Figs. 4–6). Thus in the example of Fig.
4 (C and D), EPSP amplitude increased from 1.90 mV to a
maximum of 2.95 mV but modulation decreased only slightly
(from 1.02 to 0.83). Unexpectedly, a small but significant
decrease ( 011 { 6%, n Å 8) in potentiation of the initial
EPSP (i.e., potentiation at 2 s) was observed after CPG-35348
administration (see DISCUSSION ).
To better quantify the relationship between amplitude and
modulation, we plotted the change in amplitude modulation
as a function of the change in EPSP amplitude for individual
motoneurons in baclofen- and CPG-35348-treated rats (Fig.
7). Each straight line represents the least squares fit of the
points in an individual experiment obtained after the drug
was administered (as shown in Fig. 6, as a function of time).
For the 12 connections studied with baclofen, 2–14 measure-
FIG . 3. Modulation (Eq. 1; A), potentiation (Eq. 2; B), and facilitationdepression (Eq. 3; C) as functions of EPSP18 Hz amplitude in untreated rats.
Each point represents data obtained from a single motoneuron. ( – – – ),
), linear regressions. r Å correlation coefficient
ratio value Å 1.0; (
for the regression.
mitter release pharmacologically on EPSP amplitude and
EPSP amplitude modulation. In 12 motoneurons, baclofen
was administered iv while the motoneuron penetration was
maintained and EPSP amplitude, amplitude modulation, and
potentiation were measured during the period of EPSP amplitude decline. An example of this is shown in Fig. 4, A and
B. Note that during the 45 min over which the motoneuron
penetration was maintained, EPSP amplitude decreased from
0.43 mV to 0.11 mV and EPSP amplitude modulation ratio
increased concomitantly from 0.86 to 1.77. In addition, the
potentiation ratio decreased from 1.4 to 1.18 ( 016%). Similar data were obtained at all connections studied in this way
(Fig. 5). On average (n Å 12), baclofen produced maximum
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FIG . 4. Ia-motoneuron EPSPs generated in a MG motoneuron in response to 18-Hz train stimulation and 167-Hz burst stimulation of LGS
nerve before (A) and after (B) baclofen administration and before (C) and
after (D) CGP-35348 administration. Note that baclofen administration
produces a decrease in EPSP18 Hz amplitude and a positive shift in amplitude
modulation during high-frequency stimulation, whereas the opposite occurs
after CGP-35348.
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185
the population of medial gastrocnemius motoneurons was
markedly different ( 00.08, n Å 103).
The shape of the EPSP was examined before and after
administration of baclofen (n Å 8) and CGP-35348 (n Å
7). After baclofen administration, no change was observed
in the shape of the EPSP in a majority of the cells (5/8).
However, faster decay of the EPSP was detected in one cell
and, at two others, the rise time of the EPSPs appeared to
be faster. After CGP-35348 administration, there was no
change in the time course or shape of the EPSPs in five of the
seven cells examined. One of the remaining cells exhibited a
faster EPSP decay whereas the other one exhibited a slower
EPSP decay.
Peak potentiation
FIG . 5. Histograms of EPSP18 Hz amplitude (A), modulation (B), and
potentiation (C) in untreated rats (top) and after baclofen (middle) or
CGP-35348 (bottom) administration. Control group (n Å 103) was either
untreated rats or ones that had been injected with saline. Values after
baclofen (n Å 32) were steady-state values from cells ( n Å 12) followed
for several hours immediately after baclofen was administered and values
obtained from other cells (n Å 20) impaled up to 3.3 h after baclofen had
been given. In case of CGP-35348 only 8 cells studied over course of
administration of this drug are shown because unlike effects of baclofen,
effects of CGP-35348 appeared to be short-lived. Histograms document
smaller values of EPSP amplitude and potentiation and more positive values
of modulation after baclofen and larger values of EPSP amplitude, slightly
more negative (less positive) values of modulation and identical values of
potentiation after CGP-35348.
ments were made at individual connections after administration of baclofen. Because of the small number of measurements in some cases or because the rapid decrease in amplitude and increase in amplitude modulation caused many of
the points to be clustered, the data at only six connections
exhibited a significant correlation. However, all 12 are
shown to demonstrate the similarity in the results. For CGP35348 experiments, 5–23 measurements were made per cell
and the plots revealed qualitatively similar results although
the slope was substantially smaller than in the case of baclofen. Five of the eight experiments yielded a significant correlation. It should be noted that the EPSP amplitude before
drug treatments was similar in all these experiments and
became smaller after baclofen and larger after CGP-35348
with corresponding changes in the modulation values.
We have previously demonstrated a semilogarithmic relationship between modulation and EPSP amplitude for Ia
EPSPs in cat medial gastrocnemius motoneurons (Collins et
al. 1988). We replotted the data for the subset of experiments
with significant correlations displayed in Fig. 7, using ‘‘ln
amplitude’’ as abcissa (i.e., semilog plot; Fig. 8). Although
the effect of CGP-35348 on modulation is much smaller
than that seen with baclofen (Fig. 7), the slopes of the
semilogarithmic regression lines were similar. In the case of
baclofen-treated motoneurons, the slopes of the regression
lines between modulation and ln EPSP amplitude ranged
from 00.29 to 00.63 (mean 00.55 { 0.14, n Å 6). The
slopes of the corresponding regression lines for motoneurons
studied with CGP-35348 ranged from 00.22 to 00.42 (mean
00.36 { 0.08, n Å 5). However, the slope of the regression
line for modulation as a function of EPSP amplitude across
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As pointed out above, application of baclofen or CGP35348 generally reduced potentiation of the first EPSP in
the burst evoked 2 s after the preceding burst. In previous
work in the cat, we demonstrated that peak potentiation after
the burst is reached 50–100 ms after the burst and it then
undergoes a decrease (Davis et al. 1985, 1987). In a small
subset of cells studied here, we examined potentiation 50–
100 ms after the burst and found that it was generally greater
than that seen 2 s after the burst. During baclofen administration, peak potentiation declined, whereas after CGP-35348
it was unaffected.
FIG . 6. Plots of time course of changes in EPSP18 Hz amplitude, modulation, and potentiation after baclofen (A) and CGP-35348 (B) administration
in two different motoneurons in different preparations. Note opposite effects
of these agents on EPSP amplitude and modulation and nonreciprocal effects on potentiation.
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Effects of baclofen and CGP-35348 on motoneuron
properties
To assess the possibility that baclofen or CGP-35348 may
influence EPSP amplitude via a postsynaptic action, motoneuron input resistance and rheobase were measured before
and after drug administration. In addition, repeated measurements were obtained from motoneurons in the absence of
drug administration. In no case was a significant change
(paired sample t-test) in motoneuron input resistance or
rheobase detected after drug administration or with repeated
measurements. Before and after baclofen treatment, mean
motoneuron input resistances were 1.53 and 1.67 MV, respectively (9.8%, P ú 0.1, n Å 10), and mean rheobase
values were 8.66 and 8.63 nA, respectively ( 00.5%, P ú
0.9, n Å 8). After CGP-35348 treatment, an increase in
mean input resistance from 1.70 to 1.95 MV (13.5%) was
noted, which was not statistically significant (P ú 0.05,
n Å 9) and mean rheobase was unaffected (5.73–5.72 nA,
00.2%, P ú 0.9, n Å 9). Similar repeated measurements
over approximately the same time period without drug administration revealed no significant change in mean input
resistance (1.86–1.94 MV, 4.3%, P ú 0.6, n Å 16) or in
FIG . 8. Plot of regression lines of modulation versus ln EPSP18 Hz amplitude for individual motoneurons after baclofen (dark arrows) or CGP-35348
(shaded arrows) administration. Each arrow is regression through several
measurements of EPSP18 Hz amplitude and modulation acquired at different
times after drug administration (as in Fig. 6) and arrow indicates direction
of change. Only cells for which regression line was significant (P õ 0.05)
are displayed here. EPSPs from cells treated with baclofen or CGP-35348
exhibited similar slopes when plotted on semilog coordinates. Thin line is
regression through similar data obtained from 103 motoneurons in untreated
rats. Note that regression lines from baclofen- and CGP-35348-treated motoneurons have similar slopes which are markedly different from slope of
regression line through population of motoneurons in untreated rats.
mean rheobase (7.07–6.76 nA, 04.4%, P ú 0.4, n Å 33).
Activation of GABAB receptors has been reported to hyperpolarize hippocampal cells via an increased K conductance
(Dutar and Nicoll 1988; Gahwiler and Brown 1985). However, no consistent change in motoneuron membrane potential was observed after baclofen or CGP- 35348, either directly by measurements of membrane potential or indirectly
by comparison of action-potential amplitude before and after
application of these drugs. Together, these results are consistent with those from previous studies and indicate that the
actions of baclofen in the Ia-motoneuron system are exclusively presynaptic, and suggest the same for CGP-35348,
although more direct tests are required.
DISCUSSION
FIG . 7. Each line is linear regression fit to values of EPSP amplitude
and EPSP amplitude modulation values for each of 12 connections studied
for up to 2.8 h after baclofen administration (an example of which is
displayed as a function of time in Fig. 6A) and 8 connections in rats treated
with CGP-35348 (Fig. 6B). Note that slope of changes after CGP-35348
is less negative than after baclofen. Thin line is regression line for EPSPs
evoked in 103 cells in untreated animals.
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In this study, Ia-motoneuron EPSP amplitude was altered
by pharmacologically decreasing or increasing the probability of transmitter release and the resultant changes in EPSP
amplitude modulation and potentiation were observed. A
major question was whether or not the correlated changes
in these synaptic variables measured in a single motoneuron
would match those measured across the different motoneurons in the pool. Such an observation would strengthen the
inference that probability of release of transmitter differs
systematically in terminals on motoneurons across the pool
according to the amplitude of the EPSP they produce.
Baclofen was used to diminish probability of transmitter
release because previous studies indicate that the baclofeninduced decrease in EPSP amplitude at the Ia-motoneuron
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AMPLITUDE MODULATION AT THE IA-MOTONEURON SYNAPSE
synapse results from an exclusively presynaptic action (Edwards et al. 1989; Lev-Tov et al. 1988; Peng and Frank
1989). This is a GABAB receptor-mediated, G-protein
linked, direct effect on voltage-sensitive Ca 2/ channels (i.e.,
not through second messengers) leading to a decrease in
Ca 2/ entry into the presynaptic terminals (Dunlap and Fischbach 1981) and a decrease in the probability of transmitter
release (Deisz and Lux 1985; Dolphin and Scott 1987).
GABAB receptors have been reported at the sites of presumed Ia-afferent terminals in receptor-binding experiments
(Bowery et al. 1987; Price et al. 1984). In the present study,
no significant change in motoneuron rheobase, input resistance, or membrane potential was observed after either
baclofen or CGP-35348 administration (see also Lev-Tov et
al. 1988), consistent with the lack of a postsynaptic effect,
although the change in decay time of some of the EPSPs
provides some challenge to the generality of that conclusion.
Quantal analysis has revealed a reduction in quantal content
rather than in quantal amplitude after baclofen administration (Edwards et al. 1989; Peng and Frank 1989), suggesting
no desensitization of postsynaptic receptors mediating the
Ia EPSP, although this may not be generally true (see Discussion section in Otis and Trussell 1996). Furthermore,
baclofen has similar effects on proximally and distally generated EPSPs suggesting no selective effect on synaptic inputs
to distal dendrites (Edwards et al. 1989). The lack of an
effect of baclofen on terminal potentials (Lev-Tov et al.
1988; Peng and Frank 1989) and on the electrical component
of mixed electrical/chemical synapses (Peng and Frank
1989; Shapovalov and Shiriaev 1982) argues against an effect on impulse invasion into the terminals. Antagonism of
GABAB receptors via CGP-35348 is also likely presynaptic,
although direct effects on postsynaptic receptor sensitivity
cannot yet be ruled out for this drug.
Baclofen administration resulted in a marked decrease in
EPSP amplitude that was coincident with a shift from negative to positive (or an increase in positive) amplitude modulation, whereas CGP-35348 produced the opposite increase
in EPSP amplitude and a negative shift in modulation. A
likely explanation for the positive shift in amplitude modulation with baclofen is diminished Ca 2/ entry into the terminals resulting in a decreased probability of transmitter release
and consequently less transmitter depletion and thus less
EPSP depression during repetitive stimulation (see INTRODUCTION ). The reduced depression revealed the underlying
tendency of synapses to exhibit facilitation resulting in more
positive (or less negative) amplitude modulation during
high-frequency Ia-afferent stimulation. A similar decrease
in EPSP amplitude and positive shift in amplitude modulation has been observed in the in vitro neonatal rat spinal
cord when extracellular Ca 2/ concentration was reduced
(Pinco and Lev-Tov 1993; Seebach and Mendell 1996).
The opposite effects on EPSP amplitude and amplitude
modulation during high-frequency stimulation would be expected to occur after CGP-35348 administration because of
an increase in Ca 2/ current in Ia terminals (release from
GABAB-induced decrease in Ca 2/ entry). The increased
Ca 2/ entry would elevate probability of transmitter release
and residual Ca 2/ , but this enhanced release would also
increase transmitter depletion, tending to mask any underlying facilitation. Thus the net effect expected is a negative
/ 9k23$$ja03
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187
shift in amplitude modulation. Substantial increases in EPSP
amplitude occurred as expected; however, these were accompanied by only relatively small changes in EPSP amplitude
modulation toward more negative values. Assuming that the
magnitude and sign of modulation depends on the balance
between residual Ca 2/ and transmitter depletion, it appears
that this balance measured as a function of the change in
EPSP amplitude is affected differently by baclofen and by
CGP-35348. These differences in the responses to the
GABAB receptor agonist and antagonist may relate to the
EPSPs being in different portions of the amplitude spectrum,
which was the result of treatments that altered Ca 2/ influx
in opposite directions. These findings are consistent with
a nonlinear relationship between probability of transmitter
release, EPSP amplitude and modulation, probably closer to
a semilogarithmic relationship (Fig. 8), as has been found
in the cat (Collins et al. 1988). The cellular basis for this
nonlinearity is presently unknown, but it is important to
recognize that transmitter release is probably not uniform at
the different release sites of a given afferent (Redman 1990;
Walmsley et al. 1988) and this could be a source of nonlinearity in these relationships.
Manipulation of GABAB receptor activity led to a much
steeper change in EPSP amplitude modulation as a function
of EPSP amplitude than was observed when these variables
were compared across the motoneuron pool. Because the
GABAB receptor controls Ca 2/ channel activity, manipulation of its activity can be assumed to affect probability of
release as confirmed by studies of quantal release variables.
Thus the finding that manipulation of probability of release
directly results in a much steeper slope for the relationship
between EPSP amplitude modulation and EPSP amplitude
than what is observed across the motoneuron pool suggests
that differences in EPSP amplitude in different motoneurons
are not the result of systematic differences in probability of
transmitter release. Rather it seems likely in the rat that most
of the differences in EPSP amplitude across the motoneuron
pool are the result of other factors such as the convergence
of afferents, number of boutons per afferent, and motoneuron
input resistance.
In the cat a much stronger correlation has been described
across the motoneuron pool between modulation and EPSP
amplitude than was observed here. One possible reason for
the differences between cat and rat may relate to differences
in the composition of the motoneuron pool whose members
have been suggested to retrogradely determine the transmitter release properties of the presynaptic terminals synapsing
on them (Davis and Murphey 1994; Koerber and Mendell
1991; Mendell et al. 1994). In the cat, type S motoneurons
make up about 25% of the medial gastrocnemius motoneuron
pool (Burke 1967), whereas in the male rat used here the
percentage of type S motoneurons has been estimated at
about 10% (Gardiner 1993). Because type S motoneurons
in the cat display the most consistent negative values of
modulation (Mendell et al. 1995), we can infer that in the
present experiments in the rat these were few in number.
Furthermore, no values of modulation ú55% were seen in
the rat in contrast to values ú75% seen regularly in the cat
at connections on type F motoneurons with small EPSPs
(Collins et al. 1988; Mendell et al. 1995). Both of these
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K. R. PESHORI, W. F. COLLINS, AND L. M. MENDELL
factors likely contribute to the shallow slope in the modulation versus amplitude relationship in the rat.
The uniform decrease in potentiation after baclofen is consistent with the mechanism of action of this agent, i.e., decreasing Ca 2/ entry into the presynaptic terminals. This does
not agree with previous reports of an increase in potentiation
after baclofen (Lev-Tov et al. 1988). However, the changes
in potentiation after baclofen in that experiment likely resulted from the removal of the profound depression that
follows the 30-s, 500-Hz train used as the conditioning burst.
The brief bursts used in the present experiments do not have
this complication (see Davis et al. 1985, 1987). It is interesting that baclofen produces a decrease in potentiation after
the burst but a tendency to exhibit more facilitation during
the burst. This implies that different mechanisms account for
the enlarged EPSPs during and after the burst. As discussed
above, the behavior during the burst may be determined by
the balance between transmitter depletion and facilitation
resulting from residual Ca 2/ , whereas for at least 2 s after
the burst the residual Ca 2/ may be dominant. Unexpectedly,
a decrease in potentiation was observed after CGP-35348
when enhanced Ca 2/ entry might have been expected to
increase it. We speculate that other mechanisms, e.g., interference with transmitter replenishment or a very efficient
Ca 2/ pump, may be operating under these conditions and
that this may also account in part for the relatively small
changes in modulation induced by CGP-35348.
The fact that CGP-35348 results in an increase in Iamotoneuron EPSP amplitude in the absence of baclofen indicates the presence of tonic GABAB-mediated presynaptic
inhibition in this system, although not excluding a possible
role for g-aminobutyric acid-A (GABAA ) mediated presynaptic inhibition. Stuart and Redman (1992) concluded that
GABA-mediated presynaptic inhibition at this synapse is
mediated via GABAA and not via GABAB receptors. However it is important to note that our observations, like those of
Peng and Frank (1989), reflect the level of tonic presynaptic
inhibition of unknown origin, whereas Stuart and Redman
(1992) induced presynaptic inhibition by a conditioning volley to the posterior biceps-semitendinosus nerve. More recently, the GABAB receptor has been reported to be involved
in the tonic component of the segmentally evoked presynaptic inhibition of the lumbar extensor monosynaptic reflexes
in the cat spinal cord in contrast to the GABAA receptor,
which is involved in the short-latency component (Curtis
and Lacey 1994). Whether or not this involves different
pathways or is the result of distinct pharmacological properties of these receptors is not presently known (see Curtis
1998).
The composite EPSPs studied in these experiments resulted from activation of populations of afferents and so the
reported values of amplitude, modulation, and potentiation
are averages. We assume that the single fiber and composite
EPSPs responded in the same manner to baclofen and CGP35348. It is known that the modulation produced by different
afferents in the same motoneuron exhibits significantly less
variability than that observed in different motoneurons in
response to stimulation of the same afferent (Koerber et
al. 1991). Furthermore, single fiber and composite EPSPs
exhibit similar values of modulation during the burst
(Koerber and Mendell 1991) and potentiation after the burst
/ 9k23$$ja03
J556-7
(Davis et al. 1985, 1987). Thus the composite EPSP is likely
to have properties similar to those of the single fiber EPSPs
of which it is composed. The possibility that transmitter
release from different sites of the same afferent differs
(Redman 1990; Walmsley et al. 1988) also emphasizes the
need to consider the present results as averages. However,
it is clear that these averages were affected in a consistent
manner by the various treatments carried out here.
In conclusion, a major objective of these experiments was
to determine whether manipulation of probability of transmitter release at connections on a given motoneuron would
cause correlated changes in EPSP amplitude and frequencydependent amplitude modulation similar to those seen across
the population of motoneurons. Qualitatively, the similarity
of these relationships is indicative of a common process.
However, the correlated changes in modulation and amplitude were much greater after pharmacological manipulation
of EPSP amplitude via CGP-35348 and particularly after
baclofen than at synapses of different amplitude across the
motoneuron pool. Assuming that the pharmacological manipulations affected mainly probability of transmitter release, this suggests that differences in this factor play only
a minor role in determining EPSP amplitude across the motoneuron pool in the rat. It remains to be seen whether these
pharmacological manipulations would suggest a more prominent role for probability of transmitter release in the determination of EPSP amplitude as has been suggested to occur
in the cat (Mendell et al. 1990).
We thank T. C. Cope for valuable comments on a draft of this manuscript.
This work was supported by National Science Foundation Grant
IBN9421695 to W. F. Collins and National Institutes of Neurological Disorders and Stroke Grants 2RO1 NS-16996 (Javitts Neuroscience Award),
RO1 NS-32264, and PO1 NS-14899 to L. M. Mendell.
Address reprint requests to L. M. Mendell.
Received 2 July 1997; accepted in final form 4 September 1997.
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