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.
Relative Contribution by GABA or Glycine to
Cl-Mediated Synaptic Transmission on Rat Hypoglossal
Motoneurons In Vitro.
J. Neurophysiol. 84: 2715-2724, 2000.
The relative contribution by
GABA and glycine to synaptic transmission of motoneurons was
investigated using an hypoglossus nucleus slice preparation from
neonatal rats. Spontaneous, miniature, or electrically evoked
postsynaptic currents (sPSCs, mPSCs, ePSCs, respectively) mediated by
glycine or GABA were recorded under whole cell voltage clamp after
blocking excitatory glutamatergic transmission with kynurenic acid. The
overall majority of Cl
-mediated sPSCs was
glycinergic, while only one-third was GABAergic; 70 ± 10% of
mPSCs were glycinergic while 22 ± 8% were GABAergic. Tetrodotoxin (TTX) application dramatically reduced the frequency (and
slightly the amplitude) of GABAergic events without changing frequency
or amplitude of glycinergic sPSCs. These results indicate that, unlike
spontaneous GABAergic transmission, glycine-mediated neurotransmission
was essentially independent of network activity. There was a consistent
difference in the kinetics of GABAergic and glycinergic responses as
GABAergic events had significantly slower rise and decay times than
glycinergic ones. Such a difference was always present whenever sPSCs,
mPSCs, or ePSCs were measured. Finally, GABAergic and glycinergic mPSCs
were differentially modulated by activation of glutamate metabotropic
receptors (mGluRs), which are abundant in the hypoglossus nucleus. In
fact, the broad-spectrum mGluR agonist
(±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (50 µM), which in control solution increased the frequency of both
GABAergic and glycinergic sPSCs, enhanced the frequency of glycinergic
mPSCs only. These results indicate that on brain stem motoneurons,
Cl
-mediated synaptic transmission is mainly due
to glycine rather than GABA and that GABAergic and glycinergic events
differ in terms of kinetics and pharmacological sensitivity to mGluR
activation or TTX.
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INTRODUCTION |
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The hypoglossus nucleus
is a brain stem structure in which more than 90% of local cells are
motoneurons (Viana et al. 1990). Hypoglossal motoneurons
are cholinergic elements (Davidoff and Schulze 1988
;
Lewis et al. 1971
) that innervate tongue muscles and are
thus important for functions such as swallowing, respiration, suckling,
and vocalization (Lowe 1980
). Disorders of hypoglossal motoneurons appear to be implicated in syndromes like sleep apnea in
man (Gauda et al. 1987
; Wiegand et al.
1991
). Studies of neonatal rat hypoglossal motoneurons have
demonstrated that these cells receive synaptic inputs of excitatory or
inhibitory nature (Aldes et al. 1988
; Berger and
Isaacson 1999
; Li et al. 1997
; O'Brien et al. 1997
; Rekling 1992
; Yang et al.
1995
). The major inhibitory neurotransmitters on motoneurons
are thought to be GABA or glycine, which operate via activation of
distinct postsynaptic receptors gating Cl
channels (for a recent review, see Rekling et al. 2000
).
GABA receptors mainly belong to the GABAA class
and are reversibly blocked by bicuculline while glycine receptors are
antagonized by strychnine (Barnard et al. 1993
;
Kushe et al. 1995
; Nistri 1983
;
Rajendra et al. 1997
). The co-existence of GABA and
glycine in this nucleus raises the question of their relative
contribution to synaptic microphysiology of motoneurons.
Electrophysiological studies carried out with the spinal cord slices
(Jonas et al. 1998
) have recently suggested that GABA
and glycine may be released by the same presynaptic fiber, a notion
that would classify these two substances as co-transmitters. On dorsal
horn interneurons, GABA is co-released with ATP (Jo and
Schlichter 1999
), indicating that the same presynaptic fiber
can control postsynaptic activity via more than one transmitter.
A recent report (O'Brien and Berger 1999) has suggested
that also on hypoglossal motoneurons
Cl
-mediated, miniature postsynaptic currents
(mPSCs) are generated by co-release of GABA and glycine from the same
presynaptic fiber. Nevertheless it is still unclear how many inhibitory
synapses can operate in this fashion and if co-release takes place also in the case of network-mediated, spontaneous events (sPSCs) and of
responses evoked by electrical stimulation of afferent inputs (ePSCs).
Furthermore it is not yet known if the GABA and glycine co-release
process might be subjected to modulation via receptors located on
presynaptic fibers. In particular, as metabotropic glutamate receptors
(mGluRs) largely influence synaptic transmission presynaptically
(Cochilla and Alford 1998
; Del Negro and Chandler 1998
; Gereau and Conn 1995
; Manzoni and
Bockaert 1995
; Nakanishi 1994
; Netzeband
et al. 1997
; Salt and Eaton 1995
; Sayer
et al. 1992
; Schoppa and Westbrook 1997
;
Schrader and Tasker 1997
; Shigemoto et al.
1996
), and they are abundant in various brain stem regions (Hay et al. 1999
), it seemed interesting to investigate
if glycinergic and GABAergic transmission were similarly sensitive to
this modulatory action.
The present study of neonatal rat hypoglossal motoneurons is aimed at
addressing to what extent glycine or GABA contributed to
Cl-mediated synaptic events, their basic
properties and sensitivity to tetrodotoxin (TTX) or to the broad
spectrum metabotropic receptor agonist
(±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid
(t-ACPD). The present observations suggest that, at least in
the case of neonatal motoneurons, the vast majority of
Cl
-mediated events were not apparently due to
co-release of GABA and glycine. These two neurotransmitters seemed to
generate kinetically distinct postsynaptic signals, perhaps caused by
differential location of their receptors.
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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% ip). 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 were prepared
following the procedure described by Viana et al.
(1994). In brief, the brain stem was isolated and placed in
modified ice-cold Krebs solution (see following text) to dissect out
the lower medulla, which was then pinned to an agar block placed inside
a Vibratome chamber (filled with ice-cold Krebs solution gassed with
95% O2-5% CO2).
Two-hundred-micrometer-thick slices were cut and transferred to an
incubation chamber for 1 h at 32°C containing continuously
oxygenated Krebs medium. The incubation temperature was later lowered
to ambient level and the slices maintained under this condition for at
least 1 h before use.
Recording and electrical stimulation
For electrophysiological experiments, brain stem slices placed
in a small recording chamber were viewed with an infrared video-camera to identify single hypoglossal motoneurons within the XII nucleus. Unless otherwise stated, all cell recordings were obtained with whole
cell patch-clamp electrodes (3-5 M DC resistance) while cells were
clamped at
70 mV holding potential
(Vh). Seal resistance was usually more
than 2 G
. After seal rupture, series resistance (5-25 M
) was
routinely monitored and compensated (usually by 30%, range 20-60%).
Voltage-pulse generation and data acquisition were performed with a PC
using pClamp 6.1 software. Currents elicited by voltage steps were
filtered at 3-10 kHz and sampled at 5-10 kHz.
For extracellular stimulation (0.2 Hz; 0.2 ms; variable intensity) 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 was adjusted to obtain 25-50% failures for 100 stimuli. Evoked synaptic currents were then stored in a PC as individual files and averaged with pCLAMP software (version 6.1) after discarding failed events.
Solutions and drugs
For slice preparation and subsequent incubation, the solution (in mM) was 130 NaCl, 3 KCl, 26 NaHCO3, 1.5 Na2HPO4, 1 CaCl2, 5 MgCl2, and 10 glucose (290-310 mOsm). For electrophysiological recordings, the extracellular control solution (in mM) was 140 NaCl, 3 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose (pH 7.4, 290-310 mOsm). Unless otherwise stated, 2 mM kynurenic acid was routinely added to block glutamatergic ionotropic currents. 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). Drugs were applied via the extracellular solution (superfused at 2-5 ml/min) for a minimum of 5-10 min to reach equilibrium conditions. Pilot experiments were performed to assess the antagonism selectivity of bicuculline or strychnine under the present conditions. On cells bathed with a solution containing 1 µM tetrodotoxin (TTX) and 2 mM kynurenic acid, bicuculline (10 µM) did not significantly (P > 0.05; n = 10 cells) reduce the amplitude of peak currents evoked by application of 100 µM glycine. Likewise, strychnine (0.4 µM) did not affect significantly the amplitude of the peak current evoked by 100 µM GABA (P > 0.05; n = 7 cells). These results demonstrate that at the concentrations used in the present study bicuculline or strychnine retained their antagonism selectivity against GABAA or glycine receptors, respectively.
The following drugs were used: kynurenic acid (Sigma), bicuculline methiodide (Sigma), strychnine hydrochloride (Sigma), GABA (Sigma), glycine (Sigma), TTX (Affiniti, UK), t-ACPD (Tocris, UK).
Data analysis
Cell input resistance (Rin) was
calculated by measuring the current response to 10- or 20-mV
hyperpolarizing pulses (from 70 mV
Vh), or from the slope of the linear
part of the I-V relation obtained by applying a slowly
rising voltage signal (ramp test). Detection of single postsynaptic
currents was done with AxoGraph 3.5 (Axon Instruments, Foster City, CA)
software that 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)
. Rise time of captured single events was
measured between 10 and 90% of peak amplitude, while for average
events it was measured between 20 and 80% of peak amplitude to
minimize errors due to any single event misalignment. Exponential
fitting of captured event decay was carried out with the Chebychev
algorithm provided by AxoGraph software. The decay of the vast majority
of pharmacologically identified glycinergic or GABAergic mPSCs was best
fitted by a monoexponential function. For a small minority of mPSCs (up
to 20% of all events) the time course of decay was multiphasic and, as
a first approximation, was fitted by a single time constant, representing the weighted average of the individual components. Sigma
Plot (Jandel Scientific, San Rafael, CA) and Clampfit (Axon Instruments) softwares were used for linear regression analysis of
experimental data. Paired or unpaired t-test was used to
assess differences in mean values; P < 0.05 was
considered as the acceptable level of statistical significance.
Immunohistochemistry
Immunohistochemical detection of neuronal choline
acetyltransferase was carried out as described by Ballerini et
al. (1999) using anti-choline acetyltransferase polyclonal
antibodies (Chemicon International, Temecula, CA). Immunocytochemical
detection of
1,
2,
2, and
3 subunits of the
GABAA receptor was performed by using polyclonal
antisera (Santa Cruz Biotechnology, Santa Cruz, CA) against a peptide
mapping the amino terminus of the
1 or
2 subunits and a
monoclonal antibody against the
2/3 subunit of the same receptor
(Mize and Butler 1997
). For this purpose, the whole
brain stem was removed and fixed in paraformaldehyde (4% in phosphate
buffer solution; PBS) for 24 h at 4°C, then in sucrose (30% in
PBS) for cryoprotection for the same period of time. Transverse
sections (20-µm thin) were cut with a sliding microtome at the level
of the lower medulla from frozen blocks of tissue and placed in wells
containing the fixative until further use. After washing with Triton
X-100 (0.2% in PBS), incubating for 30 min in
H2O2 (3% in PBS), and
further washing, free-floating sections were incubated overnight at
4°C in the solution containing primary antibodies (diluted 1:100 in
PBS containing 0.2% Triton X-100 and 10% fetal calf serum; FCS).
After washing with PBS, slices were incubated for 1 h in the
solution containing the secondary antibody (diluted 1:100 or 1:50 in
10% FCS in PBS). After further washing, sections were then incubated
at room temperature for 1 h with the ABC kit for alkaline
phosphatase, washed, and developed for 20-30 min in buffer containing
(for 10 ml): 1 ml Tris (1 M; pH 9.5), 0.2 ml NaCl (5 M), 0.5 ml
MgCl2 (1 M), 10 µl levamisol (1 M) and, just
before starting the reaction, 45 µl nitrobluetetrazolium (NBT; 75 mg
in 1 ml of 70% dimethylformamide) and 35 µl
bromocloroindolilphosphate (BCIP; 50 mg in 1 ml of 100%
dimethylformamide). The reaction was stopped with PBS and slices
mounted on gelatinized slides, dried for 30 min at 55°C, dipped in
solutions of decreasing methanol concentration (100-50-0%) and
increasing xylene strength (0-50-100%), and finally covered with coverslips.
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RESULTS |
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The database of the present study comprises 77 motoneurons with
64 ± 3 pF somatic capacitance and 290 ± 20 M input
resistance (Rin). The vast majority of
cells in this area were positively stained for choline
acetyltransferase (see Fig.
1A), and were thus identified
as motoneurons (cf. Viana et al. 1990
). While the
presence of glycine receptors on neonatal rat hypoglossal motoneurons
has previously been reported (Singer and Berger 1999
; Singer et al. 1998
), the localization of
GABAA receptors on motoneurons of the same age is
uncertain. For this reason, we investigated the immunocytochemical
presence of
1,
2, and
2-3 subunits of GABAA receptors. The
1 subunit (Fig.
1B) or the
2-3 subunits (not shown) were not detected.
Conversely, Fig. 1, C and D, shows, at low and
high power, motoneuron somata extensively labeled by the
2 subunit
antibody.
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Characteristics of GABAergic and glycinergic sPSCs on hypoglossal motoneurons
In kynurenic acid solution spontaneous synaptic activity routinely
consisted of inward currents occurring on average at 1.5 ± 0.3 Hz
(n = 19 cells; see example in Fig.
2A). Under our recording conditions (symmetrical Cl concentration across
the cell membrane and
70 mV Vh),
glycinergic as well as GABAergic events were inwardly directed. To
separate them pharmacologically, we used 10 µM bicuculline or 0.4 µM strychnine, respectively, as shown in Fig. 2A,
middle and bottom; note that strychnine was
applied after bicuculline had been washed out and synaptic events had
returned to control frequency and amplitude (data not shown). On a
random sample of 11 cells exposed to bicuculline, spontaneous currents
occurred at an average frequency of 1.3 ± 0.4 Hz, a value
(90 ± 20%) not significantly different from control. Further
addition of strychnine to the same cells in the presence of bicuculline
completely abolished any residual spontaneous activity (data not
shown), indicating that under the present conditions, spontaneous
currents were mediated by activation of glycine and GABAA receptors.
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After addition of strychnine to the control solution, spontaneous currents occurred at a frequency of 0.9 ± 0.3 Hz (n = 13 cells); this corresponded to a decrease by 69 ± 8% in control event frequency. Any residual activity was completely abolished by subsequent addition of bicuculline (data not shown). The action of strychnine could not be fully reversed even after more than 30 min washout. Figure 2B (left) exemplifies the mean time course of glycinergic or GABAergic sPSCs (obtained by averaging all events recorded from 2 cells in the presence of bicuculline or strychnine). After scaling, the GABAergic response had slower decay (and rise time) than the glycinergic one (Fig. 2B, right). Table 1 confirms that GABAergic sPSCs had average kinetics significantly slower than glycinergic ones.
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Characteristics of GABAergic or glycinergic mPSCs
Figure 3A shows sample
traces (obtained at 70 mV Vh in
control solution; see top 2 records), which comprised
spontaneously occurring inward currents (mPSCs) in the continuous
presence of TTX. These events appeared at random (at a 2.2 ± 0.5-Hz frequency) with relatively fast onset (2.7 ± 0.1 ms) and
monoexponential decay (14.3 ± 0.7; n = 22 cells).
Figure 3A (middle) shows that bicuculline reduced
(by 17%) the frequency of all mPSCs and did not abolish those with
multiphasic decay (see *) in accordance with a recent report
(Singer et al. 1998
). Subsequent addition of strychnine
(plus bicuculline; Fig. 3A, bottom) completely suppressed any synaptic activity, indicating that in kynurenic acid solution mPSCs
were due to activation of GABA and glycine receptors. Pharmacologically isolated glycinergic mPSCs occurred at 1.6 ± 0.5 Hz
(n = 19 cells) and made up 70 ± 10%
(n = 16 cells; P < 0.05) of the total
events. Figure 3B shows that there was no correlation
between glycinergic mPSC amplitude and rise time, making it unlikely
that they were merely shaped by electrotonic filtering (Soltesz
et al. 1995
; Ulrich and Lüscher 1993
).
Likewise we found no correlation between mPSC amplitude and decay time
(r = 0.11, slope = 0.09 ± 0.03 ms/pA, P < 0.005). Histograms of the amplitude of glycinergic
mPSCs displayed a skewed distribution as shown in Fig. 3C
(see also Singer and Berger 1999
). In accordance with
the stochastic nature of the underlying releasing process (Fatt
and Katz 1952
), glycinergic mPSCs occurred at random as
indicated by the histogram of the inter-event intervals (Fig.
3D, in which their distribution is well fitted by a single
exponential). As shown in Fig. 3E, glycinergic mPSCs
reversed at
3 ± 5 mV, a value close to the one predicted for
Cl
-mediated currents.
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Likewise, GABAergic mPSCs were pharmacologically isolated by adding 0.4 µM strychnine to kynurenic acid and 1 µM TTX containing solution
(Fig. 4A). Subsequent
application of bicuculline blocked any residual activity. Note that on
four cells 0.4 µM strychnine fully abolished all synaptic events even
if exogenous application of GABA (100 µM) still elicited inward
currents as large as in control solution (98 ± 5%; data not
shown). Whenever present, pharmacologically isolated GABAergic mPSCs
occurred at 0.5 ± 0.2 Hz (n = 13 cells) and
represented 22 ± 8% (n = 7 cells;
P < 0.05) of all mPSCs. The large majority of
GABAergic mPSCs decayed monoexponentially as only up to 15% of them
had complex kinetics of decay. The amplitude of GABAergic mPSCs was not
correlated to their rise time (Fig. 4B) or decay
(r = 0.34, slope =
0.18 ± 0.03 ms/pA,
P < 0.0001) and was distributed in a skewed fashion
(Fig. 4C). The inter-event distribution could be
fitted with a monoexponential function to indicate the stochastic
occurrence of such events (Fig. 4D). Figure 4E
shows that GABAergic mPSCs reversed at
2 ± 1 mV in accordance with the calculated reversal potential for Cl
.
Figure 5A shows the average
kinetics of glycinergic and GABAergic mPSCs, which differed
significantly in rise time, amplitude, and decay (see Table
2 for full description of data). Figure
5B compares histograms for the mPSC decay in control
solution and after pharmacological application of bicuculline or
strychnine to dissect mPSCs into glycinergic or GABAergic ones. The
control histogram and the one for the glycinergic mPSCs appear similar
because glycinergic responses represent the large majority of events.
However, the cumulative plots for control, glycinergic or GABAergic
event decay were significantly different (P < 0.0001;
Kolmogorov-Smirnov test; not shown).
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The experiments reported so far relied on protocols in which TTX was applied from the beginning of the recording session. When we first isolated pharmacologically GABA- or glycine-mediated PSCs and then applied TTX, a major difference between GABA- and glycine-mediated transmission became apparent. As indicated by the histograms of Fig. 5C, pharmacologically isolated glycinergic events did not change in frequency or amplitude after adding TTX. This finding indicates that spontaneous glycine-mediated neurotransmission was essentially independent of network activity. Conversely, GABAergic events were slightly, yet significantly, reduced in amplitude and dramatically decreased (by more than two-thirds) in frequency, outlining the requirement for strong network activity to express spontaneous GABAergic events.
Characteristics of GABAergic or glycinergic ePSCs
ePSCs were evoked by electrical stimuli applied to the lateral
reticular formation (ipsilateral to the patched motoneuron) (see
Borke et al. 1983; Travers and Norgren
1983
; Umemiya and Berger 1995
). Since ePSCs
appeared with a relatively short, constant latency after applying weak
pulses, it seems probable that these were mainly monosynaptic events.
Further support for this notion was obtained in experiments like the
one shown in Fig. 6, A and B, in which step-wise increments in ePSC amplitude were
observed whenever the stimulus strength was increased from one range of intensity to the next. Within each stimulus range the synaptic response
remained constant, indicating that there was no gradual recruitment of
additional fibers. The constant amplitude of synaptic currents for each
range of stimulus intensity is plotted in Fig. 6B. Using the
same pharmacological antagonists employed for testing spontaneous
events, we investigated, in isolation, glycinergic (Fig.
7A) or GABAergic evoked
currents (Fig. 7B). For both types of ePSC, the calculated
reversal potential was 10 ± 3 mV (Fig. 7, C and
D). ePSCs had average rise and decay times (Fig.
7E) similar to those of sPSCs and mPSCs. These data thus
confirmed the comparatively slow kinetics of all GABAergic synaptic
currents.
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Although GABA- or glycine-mediated currents were kinetically distinct,
it seemed interesting to find out whether, on the same cell, they might
be evoked by the same electrical stimulus and what relative
contribution each component might make to the composite Cl-mediated current. The example of Fig.
7F shows a cell in which average bicuculline- or
strychnine-sensitive (after bicuculline washout) evoked responses were
recorded in kynurenic acid solution. Co-application of these
antagonists fully blocked any evoked response. Digital summation of
these two components gave a waveform that could be almost completely
superimposed to the control event in kynurenic acid solution (see Fig.
7F, bottom). On this same cell, the percentage of failure
was virtually the same in control solution (42%) or in the presence of
bicuculline (43%) or strychnine (50%). These characteristics are
compatible with a co-release mechanism of GABA and glycine but were
observed in two cells only out of a sample of eight cells that all
exhibited distinct evoked GABAergic and glycinergic components. On
seven additional cells, we observed exclusively one type of
electrically evoked events (glycinergic on 5 cells and GABAergic on 2 cells). In summary then, co-release properties of electrically evoked
transmission could be found in less than 20% of recorded motoneurons.
Modulation of sPSCs and mPSCs by mGluR activity
The broad-spectrum mGluR agonist t-ACPD was tested on
18 cells on which it evoked a slowly rising, persistent inward current (on average 20 ± 1 pA) together with a large increase in input resistance (30 ± 10%) and in synaptic activity, despite the
presence of kynurenic acid. This observation indicates that the
enhancing action of t-ACPD on synaptic activity was mainly
targeted to Cl
-mediated events. To check
whether glycinergic or GABAergic systems were differentially affected
by the activation of mGluRs, bicuculline or strychnine was added to
separate sPSC.
Figure 8A shows typical traces
of glycinergic sPSCs (in bicuculline solution) before and after
addition of t-ACPD, while Fig. 8B depicts similar
data with GABAergic sPSCs. In both cases, there was a large increase in
sPSC frequency. These results are quantified (Fig. 8C) in
terms of changes in normalized frequency or amplitude for glycinergic
() and GABAergic (
) events before and after t-ACPD.
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We next studied the effect of t-ACPD on mPSC pharmacologically separated as above. As shown by the representative traces in Fig. 9A, the frequency of glycinergic mPSCs was enhanced in a reversible fashion. On the other hand, the frequency and amplitude of GABAergic mPSCs remained insensitive to t-ACPD (Fig. 9B). The histograms of Fig. 9C provide statistical analysis of average data, confirming that the process of GABA release from pharmacologically isolated GABAergic terminals in the presence of TTX was unaffected by t-ACPD while the comparable process of glycine release was t-ACPD sensitive.
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DISCUSSION |
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The principal finding of the present study on hypoglossal motoneurons of the neonatal rat is that glycinergic synaptic events differed from GABAergic ones in terms of kinetics, frequency, and sensitivity to TTX or t-ACPD. These data do not provide evidence for substantial co-release of these transmitters at this early postnatal stage and are interpreted in terms of kinetically distinct synaptic roles for glycine and GABA.
Electrophysiological characteristics of spontaneous synaptic events
In the present investigation, synaptic currents were always
studied during pharmacological block of glutamatergic ionotropic receptors and were found to reverse near 0 mV, in accordance with the
value predicted by the Nernst equation for
Cl-mediated responses. Pharmacological
separation of glycine-mediated events from those mediated by
GABAA receptors allowed us to ascertain the
relative contribution of each transmitter to the observed activity.
Parallel experiments were carried out to establish that strychnine or
bicuculline retained their receptor selectivity at the concentrations
used in the present study. Because 0.4 µM strychnine did not affect
the inward current evoked by 100 µM GABA and 10 µM bicuculline was
ineffective against 100 µM glycine, we concluded that each antagonist
retained the expected receptor specificity of action. It should also be
noted that co-application of bicuculline and strychnine fully
suppressed any synaptic activity, indicating that the synaptic currents
measured under the present conditions were only mediated by glycine or
GABAA receptor activation.
In general, glycinergic events were significantly faster than GABAergic events and occurred at higher frequency. The effect of TTX on glycinergic or GABAergic events was dramatically different. In fact, the frequency of glycinergic events remained substantially unchanged (and their amplitude was not significantly depressed), whereas GABAergic events were reduced in amplitude and, especially, in frequency. This observation indicated that asynchronous release of GABA largely depended on network activity while the release of glycine did not.
The difference in kinetics between glycinergic and GABAergic events was also found when mPSCs were analyzed. In fact, GABAergic events were significantly slower, smaller and occurred at lower frequency than their glycinergic counterparts. Despite these differences in kinetic behavior, in control solution it was not possible to identify, within the cumulative histogram of event decay, separate components due to either glycinergic or GABAergic mPSCs because in control solution the histogram was largely shaped by glycinergic events.
In some cases 0.4 µM strychnine completely suppressed any
Cl-mediated synaptic events even if exogenously
applied GABA was still able to elicit robust inward currents. The
contribution of any tonic (action potential-independent) release of
GABA to the overall synaptic activity of resting hypoglossal
motoneurons must therefore have been quite small even though
GABAA receptors were functional on these cells
and demonstrably present at somatic level. In fact, our
immunocytochemical data indicated the presence of the
GABAA receptor
2 subunit on the cell body of
neonatal motoneurons. This subunit is a major constituent of developing GABAA receptors, while the
1 subunit, which
could not be found in the present experiments, is indeed typical of
adult GABAA receptors (Fritschy and Mohler
1995
; Fritschy et al. 1994
; Mohler et al. 1996
).
It is interesting that a small fraction of mPSCs displayed complex
decay kinetics that might have suggested simultaneous activation of
GABAA and glycine receptors by synaptic
co-release of both transmitters (Jonas et al. 1998;
O'Brien and Berger 1999
). Nevertheless subsequent
application of either bicuculline or strychnine failed to abolish this
subpopulation of mPSCs: this result suggests that a high order of mPSC
decay is not per se indicative of co-release mechanism. Complex decay
might reflect spatial segregation of synaptic receptors or
heterogeneous receptors for the same transmitters (Lewis and
Faber 1996a
,b
).
Differences between GABAergic and glycinergic inputs
The large reduction in GABAergic events brought about by acute
application of TTX suggests that, even in the absence of
glutamate-mediated synaptic transmission, GABAergic interneurons were
spontaneously active because they either received a nonglutamatergic
drive or fired spikes due to the operation of their intrinsic
conductances. When glutamatergic transmission was blocked, glycine
releasing neurons were not spontaneously active. Full elucidation of
the mechanisms underlying such a different behavior is not currently available. However, one likely explanation is that glycinergic interneurons might have had their somata severed during slice preparation. In this case, glycine release would have merely reflected spontaneous quantal discharge of this neurotransmitter, a process undoubtedly endowed with either a high probability of release or a
high-density of active synaptic sites (Singer and Berger 1999) in view of the large, frequent events detected in the
present study. Vice versa, a substantial number of functional GABAergic neurons might have been preserved in the slice preparation: their collective network behavior would have been thus responsible for their
strong sensitivity to TTX action. However, immunocytochemical studies
show that GABAergic and glycinergic premotoneurons are distributed in
several areas of the brain stem and are often not spatially segregated,
although their relative preponderance in each projection pathway is
unclear (Li et al. 1997
). Another possibility would be
that glycinergic boutons had a much higher probability of release than
GABAergic ones, either because glycinergic terminals possess a more
efficient release machinery or because locally released GABA inhibits
its own release (Lim et al. 2000
).
Electrophysiological characteristics of stimulus-evoked synaptic currents
Minimal stimulation of afferent inputs is supposed to activate one
or very few presynaptic inputs to the recorded cell (Raastad 1995). In the present experiments, this stimulation protocol
was applied to the fibers originating from the lateral reticular
formation (Umemiya and Berger 1995
). When examining GABA
or glycine mediated responses separately, their overall properties like
rise and decay times were similar to those of spontaneous PSCs mediated
by glycine or GABA, respectively.
On eight cells, recording stability was sufficiently long to allow
studying the probability of failures first in control solution, then in
the presence of bicuculline, and, finally, in the presence of
strychnine (after bicuculline washout). On two cells only an approximately equivalent number of failures was found under all three
experimental conditions, an observation that would make compatible
co-release of glycine and GABA in this limited number (less than 20%)
of cases. However, other data (differential TTX sensitivity,
persistence of complex decay mPSCs during antagonist application) did
not support the co-release hypothesis. Further clarification of this
issue might be provided by pair recording from a single presynaptic
fiber and its postsynaptic cell, a hardly achievable aim with a slice
preparation. To explore the co-release question with alternative
strategies, we tested the effect of up-regulating
Cl-dependent synaptic transmission with
t-ACDP, which is known to modulate glycine- or GABA-mediated
synaptic transmission on other neurons (Bond and Lodge
1995
; Chu and Habits 1998
; Miles and
Ponder 1993
).
Differential modulation of glycine- or GABA-mediated transmission by t-ACPD
In analogy with other studies (Dong et al. 1996;
Schoppa and Westbrook 1997
), t-ACPD induced
an inward current, associated with a large increase in the frequency of
spontaneous PSCs. The action of t-ACPD is complex and
exerted at pre and postsynaptic level via distinct receptor subclasses
(Nakanishi 1994
). It seems likely that on hypoglossal
motoneurons the effects of this substance on spontaneous events were
chiefly generated at presynaptic level since the main change was in
frequency rather than amplitude. In the case of mPSCs,
t-ACPD increased the frequency of glycinergic events only,
presumably via a presynaptic site of action. There is no current
evidence for selective up-regulation of glycine receptors by
metabotropic receptor activation at postsynaptic level. The
differential action by t-ACDP on glycinergic versus GABAergic mPSCs suggests that the action of this substance was not a
mere epiphenomenon of a rise in cell input resistance that enabled
detection of electrotonically remote events.
The present observations also help to clarify the question of co-release of GABA and glycine from the same presynaptic cell. The fact that t-ACPD enhanced glycine release without affecting GABA release in TTX solution suggests that co-release did not take place. Co-release might have taken place in limited instances when some inputs were electrically stimulated although another possibility is that even "minimal stimuli" might have activated a larger number of fibers than anticipated. In the latter case, summation and/or occlusion of inputs would have been likely. These data thus suggest that caution is necessary when considering the possibility of co-release on the basis of minimal stimulation experiments in which unequivocal control over presynaptic inputs is lacking.
Can differences in kinetics between glycinergic and GABAergic events be due to spatial segregation of their receptors?
The time course of GABAergic sPSCs, mPSCs, and ePSCs was always
slower than the one of glycine-mediated responses. The slow decay of
GABAA-mediated events (see also Banks and
Pearce 2000; Banks et al. 1998
; Rossi and
Hamman 1998
) was unlikely due to electrotonic filtering of
remotely generated responses as there was no apparent correlation
between amplitude and rise time (or decay) of GABAergic events
(Soltesz et al. 1995
; Ulrich and Lüscher 1993
). Although the synaptic location of these receptors on
hypoglossal motoneurons has not yet been proven with ultrastructural
studies, GABAA receptors containing the
2
subunit (essential to confer them functional properties)
(Fritschy et al. 1997
) were readily found on the soma of
these cells. The same subunit has also been detected on motoneuron soma
in the adult rat (Fritschy and Mohler 1995
). Likewise,
glycine receptors are found on the cell body of neonatal (Singer
et al. 1998
) and adult (Racca et al. 1998
) hypoglossal motoneurons. These results collectively suggest that GABA
and glycine receptors have dendritic as well as somatic location. In
light of these considerations, it seems that the slow kinetics of
GABAergic events perhaps require a different interpretation.
It seems unlikely that some intrinsic properties of
GABAA receptors were responsible for the slow
kinetics as 2-subunit-containing receptors generate fast onset
responses (Lavoie et al. 1997
; McCellan and
Twyman 1999
). Another possibility is that
GABAA receptors spread out beyond the subsynaptic
area and were thus activated by transmitter spillover (Barbour
and Hausser 1997
; Brickley et al. 1996
;
Faber and Korn 1988
; Isaacson et al.
1993
; Kullmann and Asztely 1998
; Rossi
and Hamman 1998
). The slow rise time of GABA-mediated events
might then represent the time integral required to activate a sparse
population of receptors (Clements 1996
; Jones et
al. 1998
; Kruk et al. 1997
; Maconochie et
al. 1994
; Uteshev and Pennefather 1996
, 1997
).
In conjunction with this hypothesis, the small amplitude of GABA events
may indicate a low density of subsynaptic GABAA receptors.
This proposal is in accordance with the mechanisms of GABAergic
transmission operation in the dorsal horn of the spinal cord (Chery and de Konick 1999) or in the hippocampus
(Banks and Pearce 2000
; Banks et al.
1998
), where most GABAA receptors are
extrasynaptic and are activated mainly by GABA spillover. The en
passant varicosities made by GABAergic fibers on hypoglossal
motoneurons (Takasu and Hashimoto 1988
) might represent
the structures involved in this form of synaptic transmission in the
hypoglossus nucleus.
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ACKNOWLEDGMENTS |
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We thank Drs. Simona Capsoni and Massimo Righi for help with histological and immunocytochemical experiments.
This work was supported by Istituto Nazionale di Fisica della Materia and by Ministero dell'Universita' e della Ricerca Scientifica e Tecnologica.
Present address of R. Donato: Div. Neuroscience, Baylor College of Medicine, Houston, TX 77030.
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FOOTNOTES |
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Address for reprint requests: A. Nistri, Biophysics Sector and Istituto Nazionale di Fisica della Materia Unit, International School for Advanced Studies (SISSA), Via Beirut 4, 34014 Trieste, Italy (E-mail: nistri{at}sissa.it).
Received 24 April 2000; accepted in final form 10 August 2000.
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REFERENCES |
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