Biophysics Sector and Istituto Nazionale di Fisica della Materia Unit, International School for Advanced Studies (SISSA), 34014 Trieste, Italy
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ABSTRACT |
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Donato, Roberta and
Andrea Nistri.
Differential Short-Term Changes in GABAergic or Glycinergic
Synaptic Efficacy on Rat Hypoglossal Motoneurons.
J. Neurophysiol. 86: 565-574, 2001.
Using whole cell
patch-clamp recording from hypoglossal motoneurons of a neonatal rat
brain slice preparation, we investigated short-term changes in synaptic
transmission mediated by GABA or glycine. In 1.5 mM extracellular
Ca2+
[Ca2+]o,
pharmacologically isolated GABAergic or glycinergic currents were
elicited by electrical stimulation of the reticular formation. At low
stimulation frequency, glycinergic currents were larger and faster than
GABAergic ones. GABAergic currents were strongly facilitated by pulse
trains at 5 or 10 Hz without apparent depression. This phenomenon
persisted after pharmacological block of GABAB receptors. Glycinergic currents were comparatively much less enhanced than GABAergic currents. One possible mechanism to account for this
difference is that GABAergic currents decayed so slowly that consecutive responses summated over an incrementing baseline. However,
while synaptic summation appeared at 10-Hz stimulation, at 5 Hz
strong facilitation developed with minimal summation of GABA-mediated
currents. Glycinergic currents decayed so fast that summation was
minimal. As [Ca2+]o is
known to shape short-term synaptic changes, we examined if varying
[Ca2+]o could
differentially affect facilitation of GABA- or glycine-operated synapses. With 5 mM
[Ca2+]o, the frequency of
spontaneous GABAergic or glycinergic currents appeared much higher but
GABAergic current facilitation was blocked (and replaced by
depression), whereas glycinergic currents remained slightly
facilitated. [Ca2+]o
manipulation thus brought about distinct processes responsible for
facilitation of GABAergic or glycinergic transmission. Our data
therefore demonstrate an unexpectedly robust, short-term increase in
the efficiency of GABAergic synapses that can become at least as
effective as glycinergic synapses.
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INTRODUCTION |
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GABA and glycine are neutral
amino acids that in the mammalian CNS, in addition to their metabolic
role, operate as neurotransmitters via activation of distinct
postsynaptic receptors gating Cl permeable
channels. Glycine is mainly present in the spinal cord and brain stem,
while GABA is prevalent in higher regions of the neuraxis. In brain
stem cranial nuclei, such as, for instance, the hypoglossus nucleus,
both inhibitory neurotransmitters are present. The hypoglossus nucleus
is made up by motoneurons that innervate tongue muscles and are thus
essential for functions such as swallowing, suckling, and vocalization
(Lowe 1980
) and are involved also in maintaining the
upper airways patent. A large part of GABAergic or glycinergic inputs
to hypoglossal motoneurons originates from the reticular formation
(Li et al. 1996
, 1997
), and the respiratory centers (the
pre-Bötzinger complex in the ventrolateral medulla) (Paton
and Richter 1995
).
The co-existence of GABA and glycine in the hypoglossal nucleus
raises the question of their relative contribution to synaptic microphysiology, their interplay as transmitters, and their
differential role at functional level. Although GABA and glycine have
been proposed to be co-released at certain synapses (Jonas et
al. 1998; O'Brien and Berger 1999
), this
phenomenon does not appear frequently in the hypoglossal nucleus
(Donato and Nistri 2000
). One striking feature of
GABAergic currents (miniature, spontaneous, or evoked by electrical
stimulation) recorded from hypoglossal motoneurons of the neonatal rat
is that they are approximately two times slower than their glycinergic
counterparts (Donato and Nistri 2000
).
We wondered whether such different kinetics could lead to distinct changes in short-term efficacy of GABAergic over glycinergic transmission. This seems to be an interesting issue because during normal neuronal activity, synapses are presumably subjected to phasic and tonic signaling and might employ discrete variations in synaptic efficacy to integrate information. In principle, this might mean that the balance between GABA or glycine in information transfer at motoneuron level is a nonstationary process subjected to changes in the relative contribution by each transmitter to cell excitability.
The present study tested these possibilities by examining if the efficacy of synaptic responses mediated by GABA or glycine during high-frequency stimulation could differentially and rapidly change.
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METHODS |
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Slice preparation
Experiments were carried out on brain stem slices obtained from
0- to 5-day-old Wistar rats terminally anesthetized with 0.2 ml urethan
(10%; intraperitoneal injection). The entire procedure (including
animal handling and care) is in accordance with the Animal Welfare Act
and was approved by the Local Authority Veterinary Service. Thin brain
stem slices (200 µm thick) were prepared following the procedure
described by Viana et al. (1994) and Donato and Nistri (2000)
.
Electrophysiological recording
Single hypoglossal motoneurons within the XII nucleus were
viewed with an infrared video camera and voltage clamped with whole cell patch-clamp electrodes (3-5 M DC resistance) at
70-mV
holding potential (Vh). Series
resistance (5-25 M
) was routinely monitored and compensated
(usually by 30%, range 20-60%). Voltage and current pulse generation
and data acquisition were performed with a PC running pClamp 6.1 software (Axon Instruments, Foster City, CA). Currents elicited by
voltage steps were low-pass filtered at 3-10 kHz and sampled at 5-10 kHz.
Electrical stimulation and data analysis
For extracellular stimulation of GABAergic or glycinergic cells,
a single bipolar tungsten electrode was placed in the lateral reticular
formation. After stabilization of the synaptic response, stimulus
intensity for low-frequency pulses (0.5 Hz; 0.2 ms) was adjusted to
obtain 25-50% failures for 100 stimuli. After discarding no-response
trials, synaptic currents evoked by such low-frequency pulses were
stored as individual files and averaged (see also Donato and
Nistri 2000) to be used as reference for subsequent tests at
high-frequency stimulation. The high-frequency stimulation protocol
employed either short (4 pulses) or long (100 pulses) trains using the
same stimulus intensity that at 0.5-Hz frequency gave 25-50%
failures. Long trains consisted of 100 stimuli (range of 2- to 20-Hz
frequency) repeated at least five times (depending on the stability of
recording) at
5-min intervals (to allow for synaptic recovery). The
high-frequency (20 Hz), four-pulse protocol was repeated at
3 min
intervals for
10 times. All responses (including failures) to either
long or short trains were stored as individual files and averaged over
different trials.
Quantitative analysis of changes in synaptic currents evoked by pulse
trains was carried out with standard methods (see for example,
Seamans et al. 2001). In detail, with the long (100 pulse) train protocol, the peak amplitude of each response was
calculated from baseline current just before each stimulus artifact
(see Figs. 1A, right, and 2A, right). With the
short (4 pulse) train protocol, the peak amplitudes of the four
consecutive responses were calculated with respect to the baseline
current before the start of the train. Exponential fitting of current
decay (excluding any holding current shift during repetitive
stimulation) was carried out with the Chebychev algorhythm provided by
AxoGraph software (version 3.5; Axon Instruments) as reported earlier
(Donato and Nistri 2000
).
Using nonlinear regression analysis with minimization of sample errors,
an exponential function of the form: a + (1 a) * exp(
t/b) was fitted to the
time course of a series of evoked currents normalized versus the first
one in the train (using SigmaPlot software; Jandel Scientific, San
Rafael, CA). In this expression, a is the maximal amplitude,
b is the time constant, and t is time.
With the short-term protocol, we also measured the synaptic charge induced by GABA or glycine, namely the time integral of the synaptic current. For this purpose, the area of each response was calculated from the stimulus artifact to the next one taking as baseline an arbitrary line originating from the start of the first synaptic current.
Detection of spontaneous postsynaptic currents (sPSCs) was done
with AxoGraph software, which uses the method of minimizing the sum of
squared errors between data and a template function approximating the
width and time course of a typical synaptic event as described by
Clements and Bekkers (1997) and further detailed by
Donato and Nistri (2000)
. Unpaired t-test or
Mann-Whitney rank-sum test (SigmaStat software) was used to assess
differences in mean values; P < 0.05 was considered as
the acceptable level of statistical significance.
Solutions and drugs
For slice preparation, the solution (in mM) contained 130 NaCl,
3 KCl, 26 NaHCO3, 1.5 Na2HPO4, 1 CaCl2, 5 MgCl2, and 10 glucose (290-310 mOsm) gassed with
O2-CO2 (95/5%). For
electrophysiological recording, the extracellular control solution (in
mM) containing 140 NaCl, 3 KCl, 1.5 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose (pH 7.4, 290-310
mOsm) was continuously oxygenated and applied by superfusion (2-5
ml/min) to the slice in a recording chamber. Drugs were applied via the
extracellular solution for a minimum of 5-10 min to reach equilibrium
conditions. Kynurenic acid (2 mM) was routinely added to block
glutamatergic ionotropic currents. In accordance with our previous work
(Donato and Nistri 2000), strychnine (0.4 µM) was
applied to isolate GABAergic synaptic currents, whereas bicuculline (10 µM) was used to isolate glycinergic currents. This concentration of
bicuculline is much lower than the one inducing 50% block of the spike
late afterhyperpolarization of brain slice neurons (Debarbieux
et al. 1998
; Seutin et al. 1997
). For multiple
tests on the same cells, glycinergic transmission was studied before
the GABAergic one to exploit faster washout of bicuculline than
strychnine (Donato and Nistri 2000
). However, in view of
the extensive number of tests performed on each cell, the slow washout
of receptor antagonists, and the need to obtain closely reproducible
responses, it was not always possible to explore, on the same cell, the
differential sensitivity of glycinergic and GABAergic transmission to
various stimulus protocols. When this was precluded, data were compared
across cell groups. The patch pipette solution (in mM) was 120 CsCl, 9 NaCl, 2 MgCl2, 1 CaCl2, 10 HEPES, 10 EGTA, and 2 Mg-ATP at pH 7.2 (270-290 mOsm).
The following drugs were used: kynurenic acid (Sigma), bicuculline methiodide (Sigma), strychnine hydrochloride (Sigma), GABA (Sigma), glycine (Sigma), and 3-[[(3,4-dichlorophenyl)methyl]amino]propyl]diethoxymethyl)phosphinic acid (CGP 52432, gift from Novartis Pharma).
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RESULTS |
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High-frequency stimulation of GABAergic or glycinergic synapses
Under the present recording conditions (symmetrical
transmembrane Cl concentration and
Vh =
70 mV), synaptic currents
induced by GABA or glycine were inward. Data were collected from 27 hypoglossal motoneurons (HMs) with 270 ± 20 M
input
resistance. On neonatal HMs GABAergic synaptic currents are
consistently slower than glycinergic ones (Donato and Nistri
2000
; O'Brien and Berger 1999
). We explored whether such slow kinetics of GABA-mediated currents could be responsible for distinct changes in GABAergic synaptic efficacy following trains of electrical stimuli. For this purpose, we compared pharmacologically isolated GABAergic or glycinergic responses evoked by
repetitive stimulation (ePSCs).
Figure 1, A-G, illustrates
how we assessed the extent and time course of changes in GABAergic
transmission. In particular, raw traces (Fig. 1A, left)
demonstrate the development of facilitation of GABAergic inward
currents following repeated 5 Hz stimuli. Figure 1A
(right) exemplifies how single responses were aligned to
measure their peak amplitudes (for clarity, only the 1st 20 responses
to a single train are shown). The full time course of facilitation is
quantified in Fig. 1, B and C, which depicts the profile of changes in peak amplitude of GABAergic ePSCs evoked by
trains of 100 stimuli at 5 or 10 Hz (responses observed at each
frequency are the mean of 5 trials on the same neuron). Note that
there was a very large and gradual increment in the peak amplitude of
the inward currents that remained enhanced up to the end of the
stimulus train. We next tested if activation of
GABAB receptors by endogenous GABA might have
limited the observed increase in inward currents. To this end, tests
were repeated on the same cell in the presence of the selective
GABAB antagonist CGP-52432 (10 µM). As shown in
Fig. 1D, the extent of synaptic facilitation during the
train remained essentially unchanged. In the presence of 10 µM
bicuculline, all evoked currents were fully blocked (Fig.
1E), thus indicating that these responses were mediated by
activation of postsynaptic GABAA receptors.
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Direct data comparison between different cells under the four
experimental conditions discussed in the preceding text required normalization of the current peak amplitude (with respect to the 1st
response in the train) and extraction of objective parameters to
quantify the degree and time course of GABAergic response facilitation. Figure 1F (same cell shown in Fig. 1, A-E) shows
how we used this approach. The normalized amplitude plots indicate a
larger fractional increment at 5- than at 10-Hz stimuli and that such
facilitation could be adequately fitted by the monoexponential function
of the form f = a + (1 a) * exp(
t/b), where
a quantifies the extent of response potentiation (namely,
the fractional increase in amplitude) and b represents its
time constant. The average values for a and b are
then summarized in Fig. 1G for one cell under various
experimental conditions. Pooling data (n = 5 cells)
calculated with this method showed that facilitation of GABAergic
response amplitude at 5 Hz was many times larger than at 10 Hz but that
it was attained more slowly [for 5 Hz a = 10 ± 3 (SD) increment, b = 5 ± 2 s; for 10 Hz a = 4 ± 1 increment; b = 2.9 ± 0.7 s].
In contrast with the observations concerning GABAergic currents, glycinergic currents underwent less short-term facilitation changes even if we used tests with a broader range of stimulus frequency (2-20 Hz). Figure 2A shows raw traces of the increment in glycinergic inward currents (5-Hz stimuli). The time course of changes in peak amplitude of glycinergic ePSCs evoked by trains of 100 stimuli at 2, 5, 10, or 20 Hz on the same cell is plotted in Fig. 2, B-E. At 2 Hz (Fig. 2B), there was minimal alteration in inward current amplitude, whereas at 5 Hz (Fig. 2C), the inward current increased in a multiphasic fashion. At 10 Hz (Fig. 2D), the current amplitude increase was quite small, whereas at 20 Hz (Fig. 2E), the initial increment was not sustained. Figure 2F shows, for the same cell, the extent and time course of glycinergic response facilitation to 5 or 10 Hz fitted in the same manner as for GABAergic responses (compare with Fig. 1F). Both train stimuli induced a similar degree of glycinergic synaptic facilitation that was quantified in Fig. 2G. When we used the same analysis for four cells, the average parameters a and b were essentially the same for the 5- or 10-Hz stimulation (for 5 Hz, a = 3.4 ± 0.5 increment, b = 2.4 ± 0.7 s; for 10 Hz, a = 3.5 ± 0.6 increment, b = 1.6.4 ± 0.4 s).
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In summary then, GABAergic currents dramatically increased in amplitude with repeated stimuli while glycinergic currents were comparatively less affected.
Slow kinetics of GABAergic currents determine synaptic summation
One possible mechanism for facilitation of GABAergic
responses to high-frequency stimulation would be gradual response
summation due to the long-lasting tail of these synaptic currents
(Donato and Nistri 2000). To check this possibility, we
measured the time constant (
) of the decay of each GABAergic current
during the course of 5- or 10-Hz train stimulation. Figure
3A shows the time profile of
changes during the 100-pulse train at 5 Hz. While there was
scattering of these data points especially for the early part of the
train, there was no significant change in
value throughout the
train because these results could be fitted by linear regression with
r =
0.09, which was not significantly different from
0. The mean value of
(27.2 ± 0.8 ms, n = 5 cells; measured across the entire 5-Hz train) was slightly, though
significantly (P < 0.001), larger than the one
(24 ± 2 ms) of control responses evoked by a low-frequency (0.5 Hz) train. Figure 3C shows raw traces of individual ePSCs
(see expanded record taken at the start of the train) indicating that
baseline was often re-attained before the arrival of the next stimulus
(see also Fig. 1A). Summation of synaptic currents was
therefore an infrequent phenomenon at 5 Hz.
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Figure 3B shows, on the same cell, the scatter plot of values for GABAergic current decay at 10 Hz. Their average was
significantly larger (34 ± 2 ms, P < 0.001, n = 5 cells) than the
value at 0.5 Hz and
incremented throughout the train as confirmed by linear regression
analysis with r = 0.26, a value significantly different from 0 (P < 0.008). Inspection of the expanded trace
(Fig. 3D) indicates that there was gradual summation of
GABAergic currents for this frequency of stimulation as the tail of
each event did not fully dissipate before the subsequent one. Thus
GABAergic currents could be enhanced via two mechanisms: facilitation
(typically seen at 5 Hz) whereby individual currents peaks strongly
grew from a steady baseline and summation (at 10 Hz) when currents did
not fully dissipate before the subsequent response, which just took off
from a shifting baseline.
The phenomenon of summation is clearly demonstrated in Fig.
4A. In this example, to ensure
that pulses were delivered at a rate higher than the GABAergic current
full decay (~75 ms as decay value = 24 ± 2 ms), we
applied a short 20-Hz train. In the case of these short, high-frequency
trains, we measured peak responses in terms of total inward current
generated by GABAergic synaptic activation with respect to the baseline
level before the train. On a sample of 11 cells paired-pulse
facilitation (ratio between the 2nd and 1st response) was 2.6 ± 0.3 and grew significantly in a stepwise fashion when the ratios
between subsequent responses and the first one were considered (Fig.
4C).
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Figure 4B shows (on the same cell) that the 20-Hz short
train enhanced the second glycinergic current without much further increment for subsequent responses. Glycinergic currents were comparatively short lasting and fell to baseline before the next pulse
was delivered. Any increase in glycinergic transmission was thus not
due to summation but to facilitation. Figure 4D shows that
for an average of nine cells, the same degree of increase in amplitude
of glycinergic currents was present throughout the four-pulse train. We
also compared the cumulative increment in synaptic charge (pC) for
GABA- or glycine-mediated responses. In this way, it was possible to
analyze the temporal effectiveness of synaptic signals induced by the
two transmitters. The total synaptic charge for the cluster of four
synaptic responses was 5 ± 1 and 7 ± 2 pC for glycine and
GABA, respectively (n = 9). These results suggest that
during a very short period of high-frequency stimuli, the effectiveness
of GABA-mediated synaptic activity became very similar to
glycine-mediated one despite the fact that spontaneous glycinergic
events are faster and occur at higher frequency than their GABAergic
counterparts (Donato and Nistri 2000).
Furthermore, while enhancement of glycinergic currents by high-frequency pulses was a stereotypic process, the process for the increase in GABAergic currents relied on a dynamic mechanism, enabling progressively larger synaptic efficacy for the same synaptic input.
Role of extracellular Ca2+
It is known that several types of short-term synaptic changes are
largely dependent on extracellular Ca2+
([Ca2+]o) (for review,
see Zucker 1989). The role of
[Ca2+]o in GABAergic
synaptic facilitation was tested by changing the concentration of this
cation within the 1.5- to 5-mM range while stimulating with 5-Hz
trains. In the example of the cell shown in Fig.
5A, plots of normalized data
for the degree and time course of facilitation with 1.5, 2.5, or 5 mM
[Ca2+]o demonstrate that
only responses in 1.5 mM
[Ca2+]o underwent
sustained facilitation. The responses in 2.5 mM
[Ca2+]o had transient
facilitation, later replaced by depression. The responses in 5 mM
[Ca2+]o were unable to
show any facilitation. Similar data were collected from five cells.
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It was next examined how these changes in
[Ca2+]o might have
affected GABAergic transmission pre- and/or postsynaptically. Figure 5B shows spontaneous GABAergic currents (sPSCs) recorded in
varying [Ca2+]o from the
same cell. Changing
[Ca2+]o had a relatively
slight influence on the average peak amplitude of sPSCs because a small
increase was observed with 5 mM
[Ca2+]o only (see average
data in Fig. 5C, top). The decay time of sPSCs was not
significantly changed ( = 23 ± 1, 23 ± 1 or 26 ± 2 ms with 1.5, 2, or 5 mM
[Ca2+]o, respectively;
P > 0.05; n = 5). Conversely, the
frequency of sPSCs (expressed as inter-event interval) was strongly
enhanced with 5 mM
[Ca2+]o (Fig. 5C,
bottom). These results suggest that changes in
[Ca2+]o had a mainly
presynaptic effect on GABAergic sPSCs.
Similar experiments were carried out with glycinergic currents as shown
in Fig. 6A in which normalized
response amplitudes in 1,5, 2.5, or 5 mM
[Ca2+]o are presented. In
2.5 mM [Ca2+]o,
facilitation (normally observed with 1.5 mM
[Ca2+]o) was still
present, although reduced. In particular, the parameter a
related to synaptic amplitude went from 4.0 ± 0.1 (at 1.5 mM [Ca2+]o) to 2.01 ± 0.02, while the time course b went from 4.5 ± 0.3 to
1.8 ± 0.2 s. The latter value indicated faster attainment of steady-state facilitation level in 2.5 [Ca2+]o. With 5 mM
[Ca2+]o, there was no
apparent facilitation of glycinergic currents. Changing
[Ca2+]o had also clear
effects on spontaneous glycinergic currents as shown in Fig.
6B where amplitude and particularly frequency went up with 5 mM [Ca2+]o. Like in the
case of GABAergic sPSCs, the decay time was not significantly changed
by increasing [Ca2+]o
( = 12 ± 2, 10 ± 2, or 13 ± 1 ms,
respectively, for [Ca2+]o = 1.5, 2.5, or 5 mM; P > 0.05; n = 3).
These observations are summarized in Fig. 6C for peak
amplitude (top) and for the spontaneous inter-event
frequency (bottom). Analogous data were collected from five
cells.
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These data indicate that raising [Ca2+]o altered facilitation of evoked GABAergic transmission more strongly than facilitation of evoked glycinergic transmission. However, the sensitivity of GABAergic or glycinergic sPSCs to [Ca2+]o was similar, a fact suggesting to us that [Ca2+]o operated chiefly via a presynaptic mechanism, as the main effect was a strong change in PSC frequency coupled with slight amplitude increase, and no change in kinetics.
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DISCUSSION |
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The principal finding of the present study is the demonstration that GABAergic transmission on neonatal HMs could be strongly facilitated during high-frequency stimulation with a repertoire of changes depending on the frequency and number of electrical stimuli. On the same cells glycinergic transmission could also be facilitated but to a lesser degree and with a more stereotypic change.
Short-term changes in the efficacy of GABAergic or glycinergic synapses
During repeated stimuli, glycinergic synapses showed less
facilitation than GABAergic ones. In particular, in response to 5-Hz
stimulation, average facilitation of GABAergic peak amplitude was
10 ± 3 times, a value consistently higher than the one (3.4 ± 0.5) for glycinergic currents. Facilitation of GABAergic currents did not include activation of postsynaptic receptors other than GABAA ones because all postsynaptic responses
were eliminated by bicuculline. In other brain areas, GABA release can
be regulated by presynaptic GABAB autoreceptors
(for review, see Sivilotti and Nistri 1991). Thus, if
this situation also applied to neonatal HMs, one might have expected
that GABAB receptor activity limited the extent
of GABAergic facilitation. However, addition of CGP 52432, a potent and
selective GABAB receptor antagonist, had no significant effect on GABAergic responses (evoked at 10 Hz), indicating that GABAB receptors were either absent or
nonfunctional at this early postnatal period. This electrophysiological
observation accords with immunocytochemical studies that could not
detect the two known isoforms of GABAB receptors
in the hypoglossal nucleus, although such receptors were readily
demonstrable in adjacent areas like the nucleus of the tractus
solitarius, dorsal motor nucleus of vagus, and raphe obscurus and
magnus (McDermott et al. 1999
). Furthermore, the
GABAB agonist baclofen was ineffective on HMs at
this early postnatal age (R. Lape and R. Donato, unpublished observations). Because GABAB receptors could not
be detected histologically or functionally, it is suggested that the
comparatively less intense facilitation of glycinergic synapses was
unlikely due their preferential depression by
GABAB receptor activity.
Other receptors known to regulate synaptic transmission at presynaptic
level are metabotropic glutamate receptors (GluRs) (see Ozawa et
al. 1998 for a recent review). On neonatal HMs, GluR activity
enhances rather than depresses Cl
-mediated
synaptic transmission (Donato and Nistri 2000
). We
cannot firmly rule out that in the present study repetitive stimulation did not release glutamate to activate such receptors. Nevertheless, if
repeated stimuli had indeed released enough glutamate, this effect
should have been restricted to glycinergic terminals, where these
receptors are functional, as GABAergic terminals do not possess them
(Donato and Nistri 2000
). Yet it was GABAergic
transmission that demonstrated the largest facilitation increase. These
considerations led us to think that the difference in facilitation
observed between GABAergic and glycinergic synapses was unlikely due to
activation of mGluRs.
One further possibility to explain the difference in the extent of
facilitation between glycinergic and GABAergic synapses was that the
much slower kinetics of GABA mediated events enabled response
summation. Since the decay time constant of GABAergic events is ~100
ms (Donato and Nistri 2000), synaptic currents induced
at
10-Hz frequency of stimulation would have been long enough to
start from the tail of the preceding response. This prediction was
indeed borne out as the tails of GABAergic currents did not fully
return back to baseline at 10-Hz stimulation so as subsequent currents
emerged from a rising baseline (see Figs. 3 and 4). Nevertheless, this
phenomenon was not consistently observed with lower stimulation
frequency, implying the GABAergic facilitation did not merely involve summation.
Glycinergic currents showed less intense facilitation with minimal
evidence for response summation. Using a short train of high-frequency
(20 Hz) pulses, glycinergic current facilitation appeared fully
developed by the second response and did not further increase (longer
trains actually led to depression; see Fig. 2E). Conversely,
summation of GABAergic currents with analogous short protocols was so
strong that, in the case of a short, high-frequency train, the membrane
charge generated by GABAergic transmission was as much as the one due
to glycinergic transmission. In other words, short-term changes in the
efficacy of GABAergic synapses could compensate for the initially
slower and smaller amplitude of single GABAergic events (Donato
and Nistri 2000). Thus, short trains of GABA or glycine
synaptic signals seemed to elicit comparable synaptic charge increases.
Role for GABAA receptor desensitization?
It is somewhat surprising that, in control solution,
high-frequency stimulation did not cause depression of GABAergic
currents. GABAA receptors are known to be prone
to rapid desensitization during sustained exposure to this
neurotransmitter (Jones and Westbrook 1995, 1996
;
Jones et al. 1998
). This condition would result in
smaller response amplitude and increased decay time of the macroscopic
currents. The present data showed that the decay time of responses at 5 Hz did not significantly change during the stimulus train while their
amplitude was actually augmented. Lengthening of
values appeared
with 10-Hz trains, indicating that some desensitization of
GABAA receptors might have taken place together
with the inevitable prolongation of decay due to tail current
summation. Hence, it seems noteworthy that consecutive responses to
high-frequency stimulation (
10 Hz) did summate (see, in particular,
Fig. 4A) in contrast to the rapidly developing depression of
GABAergic transmission often observed on other brain neurons
(Sivilotti and Nistri 1991
).
Effects of [Ca2+]o on synaptic facilitation
It is known that Ca2+ is involved in the
induction of short (and long)-term changes in synaptic efficacy
(Zucker 1989). One explanation is that during intense
neuronal firing, free intracellular Ca2+ is not
fully buffered by internal stores ("residual
Ca2+ hypothesis") so as it can build up after
successive action potentials, leading to enhanced transmitter release
(Katz and Miledi 1968
; Rahamimoff and Yaari
1973
).
In our experiments on glycinergic responses to 5-Hz stimuli, we found
that with high [Ca2+]o,
facilitation, even though decreased, was still present. This finding
was accompanied by an increase in the underlying spontaneous activity
recorded at rest. Conversely, raising
[Ca2+]o blocked
facilitation of GABAergic transmission and actually led to synaptic
depression even if the frequency of GABAergic sPSCs was enhanced. On
neonatal rat motoneurons, changing
[Ca2+]o is known to
transform facilitation of excitatory synapses into depression
(Lev-Tov and Pinco 1992; Pinco and Lev-Tov
1993
): thus GABAergic transmission appeared to respond to
changes in [Ca2+]o in the
same way as other synapses do.
The mechanisms through which changes in
[Ca2+]o affected synaptic
efficacy in the present investigation are complex. On the one hand, the
higher rate of spontaneous release in high
[Ca2+]o might have
reduced the number of vesicles available for stimulus-induced release,
a factor potentially leading to synaptic depression (Bailey and
Chen 1988). On the other hand, repetitive firing induced by pulse trains might have raised free intracellular
Ca2+ to promote facilitation as indeed proposed
for hippocampal inhibitory synapses (Jensen et al.
1999
).
On HMs the observed discrepancy between the behavior of GABAergic and
glycinergic currents in high
[Ca2+]o suggests that the
pool of GABA available for transmitter release during repeated pulses
was probably more limited than the pool of glycine. This observation is
consistent with our previous study (Donato and Nistri
2000) demonstrating the much larger sensitivity of GABAergic
rather than glycinergic transmission to tetrodotoxin (TTX). Even though
the complexity and diversity of synaptic connections to hypoglossal
motoneurons make it difficult to calculate release probability
(P) with quantal analysis (see Nicholls et al.
2001
), our results imply a high release P for
glycine and a low one for GABA. It is known that the extent of synaptic
facilitation is inversely related to the value of P
(McLachlan 1978
; Stevens and Wang 1995
)
as a "low P" synapse is more likely to display
facilitation or augmentation following a repetitive stimulation,
whereas a "high P" synapse is more likely to exhibit
depression for the same stimulation. Such an inverse correlation is
possibly due to dependence of the release machinery on presynaptic
Ca2+ levels (Thomson 2000
). Hence,
it seems probable that on neonatal HMs different P values
for GABA and glycine were responsible for the dissimilar facilitation
of GABAergic and glycinergic currents.
In principle, repeated stimuli in the presence of high
[Ca2+]o might have also
increased postsynaptic Ca2+ levels to trigger
intracellular second messengers to modulate receptor sensitivity to
GABA or glycine. Some considerations make this hypothesis unlikely:
GABAA or glycinergic receptors are not permeable
to Ca2+; even if intracellular
Ca2+ was raised via influx through other channels
or via internal store release, under our experimental condition
(intracellular loading with 10 mM EGTA as buffer), postsynaptic
[Ca2+]i is supposed to be
maintained at rather constant level (Umemiya et al.
2001); raising
[Ca2+]o enhanced
glycinergic or GABAergic sPSC frequency with only slight effect on
amplitude, and no change in current kinetics.
Collectively, these results indicate that increasing [Ca2+]o mainly affected synaptic transmission at presynaptic level.
Functional significance of facilitation of GABAergic or glycinergic currents
Short-term changes in synaptic efficacy can be caused by
presynaptic changes, for example, in Ca2+entry or
sequestration, in the number of synaptic vesicles or active release
sites, or in the factors that lead to fusion of synaptic vesicles with
the plasma membrane, or in the number of functional postsynaptic
receptors (Magleby 1987). Further work will be necessary
to clarify the mechanisms involved in the facilitation process observed
on HMs. Notwithstanding the resolution of this issue, the striking
difference found in the temporal information processing of GABAergic
and glycinergic synapses opens the possibility for a markedly different
functional role for the two neurotransmitters at least as young
neonatal rat HMs are concerned. Developmental studies (Paton and
Richter 1995
) on the neonatal mouse brain stem slice
preparation (600-700 µm thick; kept at 29°C) have examined GABAergic and glycinergic inputs from respiratory neurons to HMs: whereas block of neonatal glycinergic receptors does not affect the
respiratory rhythm, block of GABA receptors increases burst duration
and cycle length of respiratory-driven rhythmic activity, and it
induces a sustained depolarization of the motoneuron membrane. Thus in
neonatal animals, GABA-mediated activity seems to have the role of
pacing the respiratory-driven rhythmic activity, whereas, at the same
stage of development, glycine seems not to be involved in regulation of
the respiratory rhythm. Since the normal respiratory rhythm of young
rodents can be
5 Hz (Jacquin et al. 1996
), it is
plausible that this frequency could physiologically enhance GABAergic
transmission to control the respiratory drive to neonatal HMs. Because
at warmer ambient temperature the GABAergic current decay kinetics are
expected to speed up, synaptic facilitation might predominate over
summation as a mechanism to boost GABAergic events.
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ACKNOWLEDGMENTS |
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This work was supported by grants from Ministero dell'Universita' e della Ricerca Scientifica e Tecnologica and from Istituto Nazionale di Fisica della Materia.
Present address of R. Donato: Div. of Neurosciences, Baylor College of Medicine, Houston, TX 77030.
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FOOTNOTES |
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Address for reprint requests: A. Nistri, International School for Advanced Studies (SISSA), Via Beirut 4, 34014 Trieste, Italy (E-mail: nistri{at}sissa.it).
Received 13 October 2000; accepted in final form 27 March 2001.
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REFERENCES |
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