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Volume 17, Number 3,
Issue of February 1, 1997
pp. 1025-1032
Copyright ©1997 Society for Neuroscience
GABAB Receptor-Mediated Inhibition of
Tetrodotoxin-Resistant GABA Release in Rodent Hippocampal CA1 Pyramidal
Cells
Wolfgang Jarolimek and
Ulrich Misgeld
Institute of Physiology, University of Heidelberg, D-69120
Heidelberg, Germany
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
Tight-seal whole-cell recordings from CA1 pyramidal cells of rodent
hippocampus were performed to study GABAB receptor-mediated inhibition of tetrodotoxin (TTX)-resistant IPSCs. IPSCs were recorded in the presence of TTX and glutamate receptor antagonists.
(R)-( )-baclofen reduced the frequency of TTX-resistant
IPSCs by a presynaptic action. The inhibition by
(R)-()-baclofen was concentration-dependent, was not
mimicked by the less effective enantiomer
(S)-(+)-baclofen, and was blocked by the
GABAB receptor antagonist CGP 55845A, suggesting a specific
effect on GABAB receptors. The inhibition persisted in the
presence of the Ca2+ channel blocker Cd2+.
There was no requirement for an activation of K+
conductances by (R)-()-baclofen, because the
inhibition of TTX-resistant IPSCs persisted in Ba2+ and
Cd2+. Because the time courses of TTX-resistant IPSCs were
not changed by (R)-()-baclofen, there was no evidence
for a selective inhibition of quantal release from a subgroup of
GABAergic terminals. (R)-()-baclofen reduced the
frequency of TTX-resistant IPSCs in guinea pigs and Wistar rats,
whereas the inhibition was much smaller in Sprague Dawley rats. In
Cd2+ and Ba2+,  -phorbol-12,13-dibutyrate and
forskolin enhanced the frequency of TTX-resistant IPSCs. Only
-phorbol-12,13-dibutyrate reduced the inhibition by
(R)-()-baclofen. We conclude that GABAB
receptors inhibit TTX-resistant GABA release through a mechanism
independent from the well known effects on Ca2+ or
K+ channels. The inhibition of quantal GABA release can be
reduced by an activator of protein kinase C.
Key words:
GABA;
baclofen;
GABAB receptors;
quantal
release;
protein kinase C;
adenylate cyclase;
presynaptic;
miniature
IPSCs
INTRODUCTION
Synaptically released GABA may activate
presynaptic GABAB receptors that reduce subsequent GABA
release. In pharmacological studies, the selective GABAB
agonist baclofen potently reduces GABA release in various preparations
(for review, see Misgeld et al., 1995 ).
Several mechanisms are presently discussed for the GABAB
receptor-mediated autoinhibition. The issue of whether inhibition of
GABA release by baclofen occurs independently of inhibition of
Ca2+ influx or activation of K+ conductance at
the nerve terminals remains controversial. So far, no effects of
baclofen on spontaneous tetrodotoxin (TTX)-resistant IPSCs have been
detected in CA1 pyramidal cells in rat hippocampal slices (Cohen et
al., 1992; Doze et al., 1995) or in CA3 pyramidal cells in organotypic
slice cultures from rat (Scanziani et al., 1992). In the latter
preparation, however, baclofen strongly reduces TTX-resistant EPSCs
(Scanziani et al., 1992). On the other hand, in cultured rat midbrain
neurons, baclofen reduces the frequency of both TTX-resistant IPSCs and
EPSCs (Jarolimek and Misgeld, 1992). Furthermore, baclofen inhibits
TTX-resistant IPSCs in thalamic slices (Ulrich and Huguenard,
1996).
A selective effect of baclofen on TTX-resistant EPSCs would suggest
that GABA and glutamate release are differentially regulated by
GABAB receptors (Thompson et al., 1993). Quantal GABA and
glutamate release are regulated by various G-protein-coupled receptors
in the hippocampus (Cohen et al., 1992; Scanziani et al., 1992, 1995; Scholz and Miller, 1992; Rekling, 1993; Thompson et al., 1993; Bijak
and Misgeld, 1995; Lupica, 1995). Acetylcholine, for example, reduces
both GABA and glutamate release (cf. Thompson et al., 1993; Scanziani
et al., 1995), suggesting that a similar modulation of release can
exist at both types of terminals. For an understanding of the
regulation of GABA release and of the functional role of presynaptic
GABAB receptors in the mammalian brain, it is important therefore to clarify the action of GABAB receptors on
quantal GABA release.
In the course of experiments on GABAB receptor-mediated
inhibition in guinea pig hippocampal slices, we surprisingly observed that (R)-()-baclofen reduced the frequency of IPSCs
recorded in TTX and hence studied this effect in more detail. Because
previous reports did not observe a baclofen-mediated inhibition of
TTX-resistant IPSCs in CA1 neurons of rats (Cohen et al., 1992; Doze et
al., 1995), we examined apparent discrepancies between our results and
previous work.
Parts of this work have been published previously in abstract form
(Jarolimek and Misgeld, 1996).
MATERIALS AND METHODS
The procedure for preparation and maintenance of hippocampal
slices followed previously described methods (Misgeld et al., 1979).
Guinea pigs (200-400 gm) were 6-9 weeks old; Wistar and Sprague
Dawley rats (20-40 gm) were 2-3 weeks old. Slices were incubated in a
solution containing (in mM): 127 NaCl, 5 KCl, 1.3 MgSO4, 1.25 KH2PO4, 2.5 CaCl2, 26 NaHCO3, 10 glucose.
Patch-clamp recordings. Whole-cell patch-clamp recordings
were made from CA1 pyramidal cells visualized through an upright microscope equipped with infrared light and differential interference optics (Stuart et al., 1993). Recordings were performed at room temperature (20-22°C). Slices were perfused at a rate of ~1.5 ml/min with a solution containing (in mM): 130 NaCl, 2 KCl,
1.3 MgSO4, 1.25 KH2PO4, 2.5 CaCl2, 26 NaHCO3, 10 glucose, pH 7.35. BaCl2 (1 mM) was used in some experiments to
block K+ conductances. To avoid precipitation,
MgCl2 was substituted for MgSO4,
KH2PO4 was omitted, and KCl was increased to
3.25 mM to keep [K+] constant. All bath
solutions were bubbled continuously with a mixture of 95%
O2/5% CO2. Recordings were obtained in the
presence of the AMPA receptor antagonist
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 µM), the
NMDA receptor antagonist CGP37849 (1 µM), and TTX (1 µM). Cd2+ was used at a concentration of 100 µM to block Ca2+ currents. In fact, voltage
steps from 65 to 0 mV did not elicit any Na+ or
Ca2+ current (n = 4). The nomenclature of
baclofen in this paper follows that used in the chemical literature
(cf. Froestl et al., 1995). The GABAB antagonist CGP
55845A, (S)-(+)-baclofen, and (R)-()-baclofen were kind gifts of Ciba Geigy, Switzerland.
(R)-()-baclofen at a concentration of 10 µM
was used throughout the study, except for the determination of
concentration-response relationships.
Recording pipettes were filled with (in mM): 130 CsCl, 10 NaCl, 0.25 CaCl2, 5 EGTA, 10 HEPES, 10 glucose, 4 Mg2+-ATP, 5 QX-314. Resistance to bath was 4-6 M before
seal formation. QX-314 was included in the patch pipette solution to
block GABAB receptor-activated K+ channels
(Nathan et al., 1990). To allow equilibration between the pipette
solution and the cell, recording of TTX-resistant IPSCs was started
10-15 min after rupturing the membrane. Once equilibrium was achieved,
even large concentrations of (R)-()-baclofen did not
activate a K+ current. Membrane currents were measured with
a discontinuous single-electrode voltage-clamp amplifier (npi, Tamm,
Germany) at a holding potential of 65 mV. All drugs were bath-applied from a wide-bore pipette used as a common outlet for different solutions. Because the pipette was positioned close to the slice and
the chamber volume was small (~400 µl), a complete exchange of the
external solution took <30 sec. All inorganic salts were of analytical
grade from Merck (Darmstadt, Germany). All drugs, except CNQX (Tocris
Cookson, Bristol, England) and TTX and QX-314 (Biotrend, Köln,
Germany), were from Sigma (Deisenhofen, Germany)
Data acquisition and analysis. Spontaneous synaptic currents
were filtered at 1.3-2.0 kHz with a 4-pole Bessel filter, sampled between 2 and 10 kHz using pClamp software (Axon Instruments, Foster
City, CA), and stored additionally on a DAT recorder. Spontaneous synaptic currents were detected by a program written in our laboratory. The program detects an event if the difference between a "running average" (data averaged for 2 msec) and data points 2 msec later exceeds a given threshold (8-14 pA). The program searches for the data
point for which the difference from the baseline is maximal, and no
further increase in the difference is detectable within a certain time
period (usually set to 2 msec). This allows the exclusion of local
maxima that arise from noise fluctuations. Events that are superimposed
but have a latency of >2 msec were detected and measured separately.
Superposition of events, however, was rare. Even in cells in which
TTX-resistant IPSCs occurred at a high frequency (7 Hz), <10% of the
events had a peak-to-peak difference of 20 msec or less
(n = 5 cells). The amplitude of an event is calculated
as the difference between the peak value (average of 1.0 msec of data
starting from the largest deflection) and the baseline (average of 2 msec of data preceding the first deflection from baseline). Each event
was inspected visually before being accepted. The 10-90% rise time
was the time difference between two data points closest in amplitude to
the value calculated by adding or subtracting 10% of the event
amplitude from the baseline or peak, respectively. The resulting list
of event amplitudes, times, and rise times was used for constructing
frequency and amplitude histograms. Events chosen for analysis of
kinetic parameters arose from baseline without an inflection during the
rising phase, and no additional event occurred during the decay phase.
For fitting model exponentials to the decay phase of spontaneous
currents, the simplex (mono-exponential) or the Chebychev
(double-exponential) algorithms of the pClamp software were used. To
decide whether a mono- or double-exponential function provided a better
fit for a given decay phase, all decay phases were fitted with a
double-exponential function. The criteria for a fit being
mono-exponential were that the faster decay time constant
( 1) differed from the slower decay time constant
(2) by <10%, or the amplitude coefficient of
2 was <15% of the coefficient of 1.
Drug effects were calculated as changes in the frequency of
TTX-resistant IPSCs. The frequency was determined from the number of
events within 60-180 sec epochs before the drug application (control),
1 or 2 min (for baclofen) or >4 min (for all other treatments) after
commencement of drug application, and 7-10 min after start of wash
(recovery). Cumulative amplitude or frequency distributions were
compared with the Kolmogoroff-Smirnoff test. Two distributions were
considered to be significantly different when p < 0.01. The paired Student's t test was used to determine statistically significant changes in kinetic parameters. Numerical values are given as mean ± SD when data are derived from a given cell. Data from several cells are presented as mean ± SEM.
RESULTS
In the presence of TTX (1 µM) and glutamate receptor
antagonists, spontaneous synaptic inward currents were recorded at a holding potential of 65 mV in CA1 pyramidal cells of guinea pig hippocampal slices (Fig. 1A). Because
of their sensitivity to the GABAA antagonists bicuculline
or picrotoxin (20 µM; n = 4), these
currents were TTX-resistant IPSCs. Bath-applied
(R)-()-baclofen reversibly inhibited TTX-resistant IPSCs
(Fig. 1A). (R)-()-baclofen (10 µM) reduced the frequency by 50% on average but did not
affect the amplitude distribution of TTX-resistant IPSCs (Fig.
1B, Table 1), suggesting a presynaptic
reduction of transmitter release. The frequency of TTX-resistant IPSCs
was reduced by (R)-()-baclofen in a
concentration-dependent manner (Fig. 1C). The
EC50 calculated from the Hill equation was 1.8 µM. Ten micromolar (R)-()-baclofen was an
almost saturating concentration (Fig. 1C) and hence was used
in all further experiments.
Fig. 1.
Effect of (R)-()-baclofen
on TTX-resistant IPSCs. A, Eight consecutive traces (2 sec each) showing TTX-resistant IPSCs before (left), 1 min after start of (R)-()-baclofen application
(middle), and 7 min after commencing wash
(right). B, Cumulative amplitude (B1) and frequency (B2) distributions are
plotted for control, (R)-()-baclofen effect, and wash.
The amplitude distributions were unchanged. The frequency distribution
was shifted to the right by (R)-()-baclofen. The
number of events used for the cumulative plots was 495 for control, 232 during (R)-()-baclofen, and 445 during wash.
C, Concentration-dependent reduction of the frequency of
TTX-resistant IPSCs by (R)-()-baclofen. Effects were
calculated as the ratio (expressed in percent) of the frequency in the
presence of a given (R)-()-baclofen concentration to
the mean frequency before and after (R)-()-baclofen
application. Squares are mean values from 5-14 cells
for each concentration. Error bars represent SEM. The solid
line is a fit (least-square method) to the Hill function,
assuming a Hill coefficient of 1.0 and a maximal reduction of the
frequency to 45% of the control. The calculated EC50 is 1.78 µM. D, Block of the
(R)-()-baclofen effect by a high-affinity GABAB antagonist (CGP 55845A). The plot of the sum of the
amplitudes sampled for 20 sec against time shows that
(R)-()-baclofen inhibited TTX-resistant IPSCs. The
effect was blocked by the GABAB antagonist CGP55845A. The
antagonist by itself had no effect. Horizontal bars
indicate the drug application periods.
[View Larger Version of this Image (36K GIF file)]
The (R)-()-baclofen-induced inhibition of TTX-resistant
IPSCs was a GABAB receptor-mediated effect. The less potent
enantiomer of baclofen (S)-(+)-baclofen (10 µM) did not reduce the frequency or amplitude of
TTX-resistant IPSCs (Table 1). Furthermore, the (R)-()-baclofen-mediated inhibition of TTX-resistant IPSCs
was fully antagonized by the high-affinity GABAB antagonist
CGP55845A (Fig. 1D, Table 1). CGP55845A itself did
not change the frequency or amplitude distributions of TTX-resistant
IPSCs (Table 1), indicating that GABAB receptors were not
activated tonically by quantal GABA release.
(R)-()-baclofen effects are independent of
Ca2+ and K+ conductances
In a recent study on rat hippocampal CA1 neurons, baclofen reduced
TTX-resistant IPSCs recorded in 20 mM extracellular
[K+] ([K+]o) but did not affect
TTX-resistant IPSCs in 5 mM [K+]o
(Doze et al., 1995). The inhibition of TTX-resistant IPSCs in high
[K+]o was likely caused by an inhibition of
Ca2+ influx into depolarized terminals, because
Cd2+ occluded the effect of baclofen. Cd2+ (100 µM) also reduced the amplitude of TTX-resistant IPSCs
recorded at low [K+]o in slices [Llano and
Gerschenfeld, 1993; Doze et al., 1995; this study (Table 1)]. In
addition, we observed an apparent reduction in frequency that may have
resulted, however, from a loss of events that became too small to
detect. To exclude an inhibition of Ca2+ influx as a
possible mechanism for a diminution of TTX-resistant IPSCs, we applied
a high concentration of Cd2+ (100 µM). Under
this condition, (R)-()-baclofen still inhibited TTX-resistant IPSCs (Fig. 2A) in all
cells (7 of 7). The inhibition was reversible on washout (Fig.
2A). As in control, (R)-()-baclofen reduced the frequency of TTX-resistant IPSCs in Cd2+,
without an effect on the amplitude distribution (Fig.
2B). The effect of (R)-()-baclofen on
TTX-resistant IPSCs was quantitatively similar in the presence and
absence of Cd2+ (Table 1). Thus,
(R)-()-baclofen reduces quantal GABA release by a
mechanism independent of Ca2+ influx through
Cd2+-sensitive Ca2+ channels.
Fig. 2.
(R)-()-baclofen-induced reduction
of the frequency of TTX-resistant IPSCs in the presence of
Cd2+. A, Display of data as in Figure
1A, B. (R)-()-baclofen reduced the frequency of TTX-resistant IPSCs in 100 µM
Cd2+. The effect of (R)-()-baclofen was
fully reversible. B, (R)-()-baclofen did not affect the amplitude of TTX-resistant IPSCs (B1)
but reduced their frequency (B2). The number of events
used for the cumulative plots was 309 for control, 126 during
(R)-()-baclofen, and 296 for recovery.
[View Larger Version of this Image (38K GIF file)]
Despite the presence of QX-314 and Cs+ in the patch pipette
so that no change in somatic K+ conductance was visible, a
K+ conductance increase in the dendrites not detected by a
somatic recording electrode might have contributed to the inhibition by (R)-()-baclofen. To exclude a postsynaptic
GABAB receptor-mediated K+ conductance
increase, the K+ channel blocker Ba2+ (1 mM) was used. Because Ba2+ is also a charge
carrier for voltage-activated Ca2+ channels,
Cd2+ (100 µM) was also applied. In the
presence of Ba2+ and Cd2+,
(R)-()-baclofen still reduced the frequency of
TTX-resistant IPSCs, and the magnitude of the reduction was similar to
the effect observed in the absence of the channel blockers (Table 1).
Again, there was no significant change in the amplitude of
TTX-resistant IPSCs (Table 1). Thus, (R)-()-baclofen does
not reduce quantal GABA release by a postsynaptic mechanism.
Inhibition of TTX-resistant IPSCs by GABAB receptors
does not change their kinetics
In CA1 neurons of rat hippocampal slices, evoked IPSCs with a slow
decay are more sensitive to baclofen than rapidly decaying IPSCs
(Pearce et al., 1995). To determine whether
(R)-()-baclofen affects only a subpopulation of
TTX-resistant IPSCs, we analyzed their kinetic parameters in the
absence and presence of (R)-()-baclofen. In rat
hippocampal CA1 neurons, the decay of electrically evoked IPSCs has
been fitted with a mono-exponential (Roepstorff and Lambert, 1994) or
double-exponential function (Pearce, 1993). In guinea pigs, a
mono-exponential function fitted well the decay phase of most
TTX-resistant IPSCs (Fig. 3A, inset). The
mean value in control solution (15.2 ± 2.0 msec; 5 cells) was
not significantly different from the mean value in the presence of
(R)-()-baclofen (16.2 ± 2.2 msec; p > 0.1) (Fig. 3A). The distribution of was unchanged by
(R)-()-baclofen, and there was no correlation between and the amplitude of individual events in the absence or presence of
(R)-()-baclofen (Fig. 3C). A detailed analysis
of the fits revealed that the decay phase of a portion of TTX-resistant
IPSCs was better fitted by a double-exponential function. The fraction of TTX-resistant IPSCs with a double-exponential decay was similar in
the presence and absence of (R)-()-baclofen (0.36 ± 0.11, 0.34 ± 0.04; n = 5). The mean values
for mono-exponential fits (control, 14.6 ± 2.0 msec;
(R)-()-baclofen, 16.0 ± 2.4 msec; p > 0.1) and 1 and 2 values for
double-exponential fits were also not significantly different (control:
1, 5.4 ± 0.5 msec, 2, 26.0 ± 2.7 msec; (R)-()-baclofen: 1, 5.8 ± 0.4 msec, 2, 26.6 ± 2.3 msec). Furthermore, the
rise time of TTX-resistant IPSCs was unaffected by
(R)-()-baclofen. The mean rise time (10-90%) in control
(1.35 ± 0.06 msec; n = 5 cells) was similar to
the value in the presence of (R)-()-baclofen (1.40 ± 0.06 msec; p > 0.1) when measured in the same cells.
Rise time distributions in both conditions were indistinguishable (Fig.
3B), and there was also no correlation between the rise time
and the amplitude of single events in the presence of
(R)-()-baclofen (Fig. 3D). The analysis of rise
time and decay time constants did not provide any evidence for the
existence of groups of TTX-resistant IPSCs with a differential
sensitivity to (R)-()-baclofen.
Fig. 3.
Kinetics of TTX-resistant IPSCs in
(R)-()-baclofen. A, Distributions of
the mono-exponential decay time constant in control solution
(mean ± SD, 21.5 ± 7.0 msec; n = 177)
and in the presence of (R)-()-baclofen (19.5 ± 6.4 msec; n = 99) were not significantly different
(p > 0.1, paired Student's
t test), whereas the frequency was reduced.
Inset shows a mono-exponential fit (time constant, ,
18.06 msec; amplitude coefficient, A, 26.8).
Calibration: 5 pA, 20 msec. White and stippled
bars are control data; black bars represent data
obtained in (R)-()-baclofen. B,
Distribution of the rise time (10-90%) of TTX-resistant IPSCs
recorded in control solution (mean ± SD, 1.63 ± 0.68 msec;
n = 267) and in the presence of
(R)-()-baclofen (1.69 ± 0.70 msec;
n = 113) were also not significantly different
(p > 0.1). C, Plot of the
amplitude of individual TTX-resistant IPSCs against their value in
the absence (C1) and presence (C2) of
(R)-()-baclofen. (R)-()-baclofen did not change the relation between the amplitude of TTX-resistant IPSC and
. D, Plot of the rise time of individual
TTX-resistant IPSCs against their peak amplitude in the absence
(D1) and presence (D2) of
(R)-()-baclofen. (R)-()-baclofen did
not change the relation between rise time and amplitude when measured
for individual events.
[View Larger Version of this Image (34K GIF file)]
Inhibition of TTX-resistant IPSCs by GABAB receptors
exists in different rodents
Because in hippocampal CA1 neurons of Sprague Dawley rats 10 µM (±)-baclofen did not inhibit TTX-resistant IPSCs in 5 mM [K+]o (Doze et al., 1995), we
first investigated possible experimental differences that could account
for the discrepancy in results. We tested a 1:1 mixture of
(S)-(+)- and (R)-()-baclofen (each 5 µM). The mixture had a smaller effect than 10 µM (R)-()-baclofen, as expected from the
concentration-response curve of the active enantiomer and the lack of
effect of the inactive enantiomer (Table 1). Second, we tested whether
a difference in species was responsible for the discrepancy. We
recorded TTX-resistant IPSCs from CA1 pyramidal cells of Sprague Dawley
rats and studied the effects of (R)-()-baclofen in the
presence of Cd2+. In contrast to the effects in guinea
pigs, 10 µM (R)-()-baclofen only slightly
reduced the frequency of TTX-resistant IPSCs in Sprague Dawley rats
(Table 1). In five of eight pyramidal cells of Sprague Dawley rats,
there was no significant reduction of the frequency of TTX-resistant
IPSCs (Fig. 4A,B). In the three cells
(from three animals) that exhibited a significant reduction, the effect
amounted to 32%. Thus, GABAB receptor-mediated inhibition may be more difficult to detect in Sprague Dawley rats than in guinea
pigs, particularly if a nonsaturating agonist concentration is used.
Sprague Dawley rats, however, are exceptional among rodents with
respect to another G-protein-mediated effect (Salin et al., 1995). To
test for species versus strain differences, we therefore recorded
TTX-resistant IPSCs in Cd2+ from another rat strain
(Wistar). (R)-()-baclofen reduced the frequency of
TTX-resistant IPSCs in hippocampal CA1 pyramidal cells of Wistar rats
(Fig. 4C,D). Again, there was no effect on the amplitude of
TTX-resistant IPSCs (Fig. 4D, Table 1). The inhibition by (R)-()-baclofen of the frequency of
TTX-resistant IPSCs in Wistar rats was significant in six of six cells
and quantitatively similar to the effect in guinea pigs (Table 1).
Fig. 4.
(R)-()-baclofen does not reduce
the frequency of TTX-resistant IPSCs in a CA1 neuron of a Sprague
Dawley rat but inhibits TTX-resistant IPSCs in a CA1 neuron of a Wistar
rat. All recordings were performed in Cd2+ (100 µM). A, Eight consecutive traces (2 sec
each) showing TTX-resistant IPSCs before (left) and 90 sec after start of the (R)-()-baclofen application
(middle), and 7 min after commencing wash
(right). B, Cumulative amplitude
(B1) and frequency (B2) distributions are
plotted for control, (R)-()-baclofen effect, and wash.
(R)-()-baclofen did not significantly reduce the
frequency or amplitude of TTX-resistant IPSCs in 100 µM
Cd2+ in this cell. The number of events used
for the cumulative plots was 180 for control, 162 during
(R)-()-baclofen, and 160 for recovery. C,
D, (R)-()-baclofen reduced the frequency of
TTX-resistant IPSCs in Wistar rats, whereas the amplitude distribution
was unchanged. The number of events used for the cumulative plots was
248 for control, 151 during (R)-()-baclofen, and 240 for recovery.
[View Larger Version of this Image (39K GIF file)]
Protein kinase C (PKC) activator reduces GABAB
receptor-mediated inhibition of quantal GABA release
In rat hippocampal CA1 pyramidal cells, activation of PKC reduces
the baclofen-induced diminution of evoked IPSCs, although the
depressant effect on the postsynaptic GABAB response is
much stronger (Pitler and Alger, 1994). Because the inhibition of
quantal release could be one mechanism by which GABAB
receptors reduce GABA release, we studied the effects of PKC activators
on the GABAB receptor-mediated inhibition of TTX-resistant
IPSCs. Cd2+ and Ba2+ were continuously present
to avoid interference with Ca2+ or K+
conductances. As reported elsewhere (Capogna et al., 1995),
-phorbol-12,13-dibutyrate (1 µM) increased the mean
frequency of TTX-resistant IPSCs not altering amplitude distribution
(Figs. 5, 6). (R)-()-baclofen reduced the
frequency of TTX-resistant IPSCs significantly less in the presence of
-phorbol-12,13-dibutyrate (Fig.
6B), indicating that activation of PKC
reduces GABAB receptor-mediated inhibition of quantal GABA
release. Forskolin (20 µM), an indirect activator of
protein kinase A (PKA), also enhanced the frequency of TTX-resistant IPSCs recorded in Cd2+ and Ba2+. After
stimulation of PKA by forskolin, (R)-()-baclofen strongly reduced the frequency of TTX-resistant IPSCs (Figs. 5C,D,
6B). Quantitatively, this effect was identical to the
effect of (R)-()-baclofen in the absence of forskolin
(Fig. 6B). On average, the increase in the frequency
of TTX-resistant IPSCs was larger with -phorbol-12,13-dibutyrate than with forskolin (Fig. 6A); however, when only
cells with similar increases in frequency were considered (compare Fig.
5; -phorbol-12,13-dibutyrate: 208 ± 22%, n = 3; forskolin: 189 ± 15%, n = 3), the effects of (R)-()-baclofen were still significantly different
(-phorbol-12,13-dibutyrate: 78 ± 8%, n = 3;
forskolin: 58 ± 10%, n = 3; p < 0.05, unpaired Student's t test).
Fig. 5.
Effects of (R)-()-baclofen on
TTX-resistant IPSCs on stimulation of -phorbol-12,13-dibutyrate
(-phorbol.) or forskolin (forskol.). TTX-resistant IPSCs were recorded in
Ba2+ (1 mM) and Cd2+ (100 µM). A, GABAB
receptor-mediated inhibition of TTX-resistant IPSCs in the presence of
-phorbol-12,13-dibutyrate. Consecutive traces (1 sec each) show that
-phorbol-12,13-dibutyrate (-phorbol.; 1 µM) strongly increased the frequency of TTX-resistant
IPSCs. (R)-()-baclofen reduced the frequency of
TTX-resistant IPSCs. B, Cumulative amplitude and
frequency distributions of TTX-resistant IPSCs reveal that neither
(R)-()-baclofen nor -phorbol-12,13-dibutyrate affected the amplitude distribution; however,
-phorbol-12,13-dibutyrate increased and
(R)-()-baclofen thereafter reduced the frequency of
TTX-resistant IPSCs. The number of events used for the cumulative plots
was 443 for control, 780 for -phorbol-12,13-dibutyrate, and 541 during -phorbol-12,13-dibutyrate and
(R)-()-baclofen. C, GABAB
receptor-mediated inhibition of TTX-resistant IPSCs in the presence of
forskolin (20 µM). Consecutive traces (1 sec each) show
that forskolin increased the frequency of TTX-resistant IPSCs. (R)-()-baclofen in the presence of forskolin still
reduced the frequency. D, Cumulative amplitude and
frequency distributions of TTX-resistant IPSCs. Neither forskolin nor
(R)-()-baclofen (traces are
superimposed) significantly affected the amplitude distribution
(D1). Forskolin increased the frequency of TTX-resistant IPSCs; (R)-()-baclofen reduced it (D2).
The number of events used for the cumulative plots was 364 for control,
652 for forskolin, and 374 during forskolin and
(R)-()-baclofen.
[View Larger Version of this Image (29K GIF file)]
Fig. 6.
Graphic representation of the effects of
-phorbol-12,13-dibutyrate, forskolin, and
(R)-()-baclofen on TTX-resistant IPSCs. All recordings
were performed in the presence of Cd2+ (100 µM) and Ba2+ (1 mM).
A, -phorbol-12,13-dibutyrate
(phorbol; 1 µM) or forskolin (forskol; 20 µM) increased the
frequency of TTX-resistant IPSCs. B, In control
(white column) and in the presence of forskolin (forskol),
(R)-()-baclofen (()Bac)
strongly reduced the frequency of TTX-resistant IPSCs. In the presence
of -phorbol-12,13-dibutyrate, the reduction was significantly
smaller (phorbol; Student's t test; p < 0.05). Columns represent
mean effect for all cells (n = 4 for
A; n = 6 for B).
Error bars represent SEM.
[View Larger Version of this Image (21K GIF file)]
DISCUSSION
Presynaptic GABAB receptors in the CA1 region of the
rodent hippocampus inhibit quantal GABA release, and this inhibition is
reduced by an activator of PKC. Inhibition of quantal release may
participate in the autoinhibition of GABA release in the hippocampus, in addition to the well-known GABAB receptor-mediated
inhibition of Ca2+ channels and activation of
K+ conductances.
Characterization of (R)-()-baclofen-induced
inhibition of quantal GABA release
(R)-()-baclofen reduced the frequency of
TTX-resistant IPSCs in CA1 pyramidal cells of the rodent hippocampus by
an action on GABAB receptors. The pharmacological
identification rests on the specific action of agonists and
antagonists. We found that the active (R)-() but not the
inactive (S)-(+) enantiomer of baclofen reduced
TTX-resistant IPSCs. Furthermore, the inhibition was blocked by a
high-affinity GABAB antagonist, CGP 55845A (Froestl et al.,
1992; Jarolimek et al., 1993). The EC50 for the
baclofen-mediated inhibition of TTX-resistant IPSCs (1.8 µM) is similar to the EC50 described for
various presynaptic baclofen effects. Typical concentrations of
baclofen used for presynaptic inhibition of IPSCs in CA1 neurons range
from 1 to 50 µM (Lambert et al., 1991; Doze et al., 1995; Pearce et al., 1995), although a high-affinity effect of baclofen (EC50 100 nM) has also been found (Pearce et
al., 1995).
Baclofen inhibits Ca2+ channels (for review, see Dolphin,
1995), and this effect may contribute substantially to the potent presynaptic inhibition of action potential-dependent GABA release in
the hippocampus (Pitler and Alger, 1994; Doze et al., 1995). In our
study on CA1 neurons of the rodent hippocampus,
(R)-()-baclofen reduced the frequency of TTX-resistant
IPSCs also after blockade of Ca2+ channels by
Cd2+. Thus, inhibition of Ca2+ channels is not
the only mechanism by which GABA can inhibit its own release. We tested
(R)-()-baclofen in the presence of both TTX and
Cd2+, because it has been shown that spontaneous synaptic
activity recorded in TTX acquires Cd2+-sensitivity when
[K+]o is raised to 20 mM in
cultured neurons (Finch et al., 1990; Jarolimek and Misgeld, 1991) and
in brain slices (Doze et al., 1995). Doze et al. (1995) found that
baclofen reduced the frequency of TTX-resistant IPSCs in 20 mM [K+]o but not in 5 mM [K+]o. Because
Cd2+ occluded the baclofen effect in 20 mM
[K+]o, they concluded that the effect of
baclofen on TTX-resistant IPSCs exclusively reflected an action on
Ca2+ channels. In cultured midbrain neurons,
(R)-()-baclofen reduced the frequency of TTX-resistant
IPSCs in both 5 and 20 mM [K+]o.
Because the effect was much stronger in 20 mM
[K+]o, we suggested that in 20 mM
[K+]o, (R)-()-baclofen acts
through an inhibition of both Ca2+ channels and quantal
release (Jarolimek and Misgeld, 1992).
Activation of a postsynaptic K+ conductance by
(R)-()-baclofen could mimic an inhibition of TTX-resistant
IPSCs; however, there was neither an
(R)-()-baclofen-induced postsynaptic K+
conductance increase nor a reduction of the amplitude of TTX-resistant IPSCs, and the inhibition of TTX-resistant IPSCs persisted in the
presence of the K+ channel blocker Ba2+ (and
Cd2+). This suggests that GABAB receptors
reduce quantal GABA release by a presynaptic, Cd2+- and
Ba2+-insensitive mechanism.
Previous studies have shown that presynaptic GABAB
receptor-mediated effects are heterogenous at different GABAergic nerve terminals in the hippocampus. A portion of evoked IPSCs is reduced only
slightly (Pearce et al., 1995) or not at all (Lambert and Wilson, 1993)
by baclofen, whereas another portion of IPSCs recorded in the same
cells is highly sensitive to baclofen. In CA1 neurons of rat
hippocampus, evoked IPSCs with a slow decay are more sensitive than
fast decaying IPSCs (Pearce et al., 1995). The analysis of the rise
time and decay time constants of TTX-resistant IPSCs in the guinea pig
hippocampus did not reveal such a divergence on the level of quantal
GABA release.
Various transmitters are known to inhibit quantal release of glutamate
and GABA (for references, see introductory remarks). The
GABAB receptor agonist baclofen was reported to inhibit
TTX-resistant EPSCs (Scanziani et al., 1992) but not TTX-resistant
IPSCs in the rat hippocampus (Cohen et al., 1992; Scanziani et al.,
1992; Doze et al., 1995). GABAB receptors, however, do
inhibit TTX-resistant IPSCs in thalamic neurons of rat slices (Ulrich
and Huguenard, 1996) and cultured rat midbrain neurons (Jarolimek and
Misgeld, 1992). This might indicate that GABAB receptor
subtypes or different second messenger systems are involved in
different brain regions of different species. The data of this study,
however, indicate that (R)-()-baclofen inhibits quantal
GABA release also in the hippocampus.
The findings of the present study contradict a previous study in which
baclofen did not inhibit TTX-resistant IPSCs in hippocampal CA1 neurons
of Sprague Dawley rats (Doze et al., 1995). We found two reasons for an
explanation of the discrepancy. We used an almost saturating
concentration of the active enantiomer of baclofen ((R)-()-baclofen) to elicit maximal effects, whereas the
less effective racemate was used in the previous study. The racemate had significantly smaller effects in guinea pig slices than the almost
saturating concentration of (R)-()-baclofen. The
baclofen-mediated reduction in the frequency of TTX-resistant IPSCs was
qualitatively similar in guinea pigs and in Wistar and Sprague Dawley
rats; however, the reduction by a saturating concentration was
quantitatively smaller in Sprague Dawley rats than in guinea pigs or
Wistar rats. The application of an agonist concentration near the
EC50 of the active enantiomer in combination with the use
of slices from "insensitive" Sprague Dawley rats together may
explain the negative finding of the previous study. In contrast to the
presynaptic inhibition of evoked EPSCs by  -opioids that is found in
hippocampal slices of various rodents but not in those of Sprague
Dawley rats (Salin et al., 1995), presynaptic inhibition of quantal
GABA release by GABAB receptors exists also in thalamic
(Ulrich and Huguenard, 1996) and hippocampal terminals of the latter
species. The inconsistency and faintness of the effect in hippocampal
terminals of Sprague Dawley rats may depend then on a different stage
of regulation of GABAB receptor-mediated inhibition among
different rodent strains, e.g., on a regulation through phosphorylating
and dephosphorylating processes.
Regulation of GABAB receptor-mediated inhibition of
quantal GABA release
The mechanism by which GABAB receptors reduce quantal
transmitter release is unknown. Many proteins that are involved in the release cascade possess potential phosphorylation sites (Südhof, 1995), which could be the target of second messengers activated by
GABAB receptors. Activation of PKA or PKC enhances quantal GABA release (Capogna et al., 1995; Sciancalepore and Cherubini, 1995;
this study), indicating that phosphorylation may play an essential role
in the tuning of quantal release. Phosphorylation by PKC not only
enhances transmitter release but also reduces the inhibition of quantal
GABA release by baclofen. In contrast, activation of PKA by forskolin
does not reduce the baclofen effect, although it also enhances
transmitter release. Thus, the autoinhibition of GABA release is
controlled by PKC in a synergistic way, i.e., a reduction in the
effectiveness of the inhibitor and an increased transmitter release. In
the CA1 region of the hippocampus, activation of PKC causes a strong
depression of the postsynaptic GABAB response and a smaller
inhibition of the baclofen-induced reduction of evoked IPSCs (Pitler
and Alger, 1994). The GABAB receptor-mediated inhibition of
IPSCs is probably caused by an inhibition of Ca2+ influx
and of quantal release. Because the GABAB receptor-mediated inhibition of Ca2+ currents may be PKC insensitive
(Diversé-Pierluissi and Dunlap, 1993), the PKC-sensitive
reduction of evoked release could be caused by the inhibition of
quantal GABA release.
Conclusion
GABAB receptors, like other G-protein-coupled
receptors, are functionally heterogenous. They can couple to
K+ channels, to Ca2+ channels, or directly to
the release cascade. For many receptors that could be characterized by
their sequence structure, the functional diversity is associated with
the existence of receptor subtypes. Receptor subtypes may be expected,
therefore, for GABAB receptors. Activation of
GABAB receptors inhibits quantal GABA release in the CA1
region of the rodent brain and is regulated by PKC. Various neurotransmitters with a G-protein requirement, including GABA, modulate quantal transmitter release (cf. introductory remarks). The
dual mode of action, i.e., modulation of voltage-gated channels and
quantal release, may represent a common feature of modulation of
transmitter release at synapses mediating fast synaptic
transmission.
FOOTNOTES
Received Nov. 14, 1996; accepted Nov. 25, 1996.
This work was supported by the Sonderforschungsbereich 317/B13 of the
Deutsche Forschungsgemeinschaft. We thank John F. X. O'Callaghan for
helpful discussions and C. Heuser and A. Lewen for excellent technical
support. The gift of baclofen, CGP55845A as well as CGP 37849, by Ciba
Geigy, Basel, Switzerland, is highly appreciated.
Correspondence should be addressed to Dr. Ulrich Misgeld, Institute of
Physiology, University of Heidelberg, Im Neuenheimer Feld 326, D-69120
Heidelberg, Germany.
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