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The Journal of Neuroscience, May 1, 1998, 18(9):3138–3146 G-Protein-Coupled Modulation of Presynaptic Calcium Currents
and Transmitter Release by a GABA Receptor

Tomoyuki Takahashi, Yoshinao Kajikawa, and Tetsuhiro Tsujimoto
Department of Neurophysiology, University of Tokyo Faculty of Medicine, Tokyo 113, Japan Presynaptic GABA receptors play a regulatory role in central (GDP␤S) abolished the effect of baclofen on both presynaptic synaptic transmission. To elucidate their underlying mechanism calcium currents and EPSCs. The nonhydrolyzable GTP analog of action, we have made whole-cell recordings of calcium and guanosine 5Ј-O-(3-thiotriphosphate) (GTP␥S) suppressed pre- potassium currents from a giant presynaptic terminal, the calyx synaptic calcium currents and occluded the effect of baclofen of Held, and EPSCs from its postsynaptic target in the medial on presynaptic calcium currents and EPSCs. Photoactivation of nucleus of the trapezoid body of rat brainstem slices. The GTP␥S induced an inward rectifying potassium current at the GABA receptor agonist baclofen suppressed EPSCs and pre- calyx of Held, whereas baclofen had no such effect. We con- synaptic calcium currents but had no effect on voltage- clude that presynaptic GABA receptors suppress transmitter dependent potassium currents. The calcium current–EPSC re- lationship measured during baclofen application was similar to that observed on reducing [Ca 2ϩ] , suggesting that the pre- synaptic inhibition generated by baclofen is caused largely by receptor; presynaptic inhibition; G- the suppression of presynaptic calcium influx. Presynaptic protein; calcium currents; inwardly rectifying potassium cur- loading of the GDP analog guanosine-5Ј-O-(2-thiodiphosphate) rents; the calyx of Held; presynaptic recording GABAB receptors are widely distributed in the presynaptic and GABAB receptors as well as metabotropic glutamate receptors postsynaptic membranes of vertebrate central neurons, and they and adenosine receptors (Barnes-Davies and Forsythe, 1995).
modulate synaptic transmission by either suppressing transmitter Here we demonstrate that the G-protein-coupled inhibition of release or hyperpolarizing postsynaptic cells (Thompson et al., calcium channels underlies the GABAB receptor-mediated pre- 1993; Kaupmann et al., 1997). At neuronal somata, GABAB receptors are known to activate G-proteins, thereby enhancing inwardly rectifying potassium channels (Andrade et al., 1986; MATERIALS AND METHODS
Sodickson and Bean, 1996) or suppressing calcium channels Preparation and solutions. Transverse slices of the superior olivary com- (Dolphin and Scott, 1987; Scholtz and Miller, 1991; Mintz and plex were prepared from 14- to 19-d-old Wistar rats killed by decapitation Bean, 1993). Compared with the wealth of information on the under halothane anesthesia. The medial nucleus of trapezoid body (MNTB) neurons and calyces were viewed with a 40ϫ, 63ϫ (Zeiss), or 60ϫ (Olympus Optical, Tokyo, Japan) water immersion lens attached to known about their presynaptic mechanism of action. In particu- an upright microscope (Axioskop, Zeiss). Each slice was perfused with lar, it is not known whether the effector of presynaptic GABAB artificial CSF (aCSF) containing 120 mM NaCl, 2.5 mM KCl, 26 mM receptors is a potassium channel (Saint et al., 1990; Thompson NaHCO3, 1.25 mM NaH2PO4, 2 mM CaCl2, 1 mM MgCl2, 10 mM and Gahwiler, 1992), a calcium channel (Scholtz and Miller, 1991; glucose, 0.5 mM myo-inositol, 2 mM sodium pyruvate, 0.5 mM ascorbic Pfrieger et al., 1994; Wu and Saggau, 1995; Dittman and Regehr, M lactic acid, pH 7.4, with 5% C O2 and 95% O2. To isolate Ca 2ϩ currents, 10 mM tetraethylammonium (TEA) chloride and 1 ␮M 1996, 1997), or exocytotic machinery downstream of calcium tetrodotoxin (TTX) were included in the aCSF. The postsynaptic patch influx (Scanziani et al., 1992; Dittman and Regehr, 1996; pipette was filled with a solution (A) containing 97.5 mM potassium Rohrbacher et al., 1997). Also, an involvement of G-proteins in gluconate, 32.5 mM KCl, 10 mM HEPES, 5 mM EGTA, and 1 mM MgCl2, pH adjusted to 7.4 with KOH. N-(2,6-diethylphenylcarbamoylmethyl)- B receptor-mediated presynaptic inhibition remains to be triethyl-ammonium bromide (QX314, 5 mM) was included in the postsyn- directly demonstrated (Thompson et al., 1993). The calyx of Held aptic pipette solution to suppress action potential generation when aCSF is an ideal preparation for directly testing these issues using did not contain TTX. For recording EPSCs, the aCSF routinely con- patch-clamp techniques (Forsythe, 1994; Borst et al., 1995; tained bicuculline (10 ␮M) and strychnine (0.5 ␮M) to block spontaneous Takahashi et al., 1996). The presynaptic terminal possesses inhibitory synaptic currents. For recording presynaptic calcium currents (IpCa), the presynaptic pipette was filled with a solution (B) containing 110 mM CsCl, 40 mM HEPES, 0.5 mM EGTA, 1 mM MgCl2, 2 mM ATP, 0.5 mM GTP, 12 mM Na2 phosphocreatinine, and 10 mM TEA, pH Received Nov. 26, 1997; revised Feb. 11, 1998; accepted Feb. 12, 1998.
adjusted to 7.4 with CsOH. Presynaptic potassium currents were re- This work was supported by the “Research for the Future” Program by The Japan corded with solution A. The presynaptic pipette solutions routinely Society for the Promotion of Sciences. We thank M. Farrant, I. D. Forsythe, T.
contained 2 mM ATP (ATP-Mg salt), 12 mM phosphocreatinine, and 0.5 Manabe, and K. Kobayashi for critically reading this manuscript. We are also mM GTP, unless noted otherwise. For paired recordings 10 mM potas- grateful to R. Y. Tsien and V. Lev-Ram for their technical advice on the caged compound photolysis system and to Novartis Pharma (Basel, Switzerland) for the sium glutamate or cesium glutamate (equimolar replacement of KCl or CsCl) was also included in the presynaptic pipette solution (Borst et al., Correspondence should be addressed to Tomoyuki Takahashi, Department of Neurophysiology, University of Tokyo Faculty of Medicine, Tokyo 113, Japan.
Recording and data analysis. Whole-cell patch-clamp recordings were Copyright 1998 Society for Neuroscience 0270-6474/98/183138-09$05.00/0 made from MNTB neurons, presynaptic calyces, or simultaneously from Takahashi et al. • Presynaptic Ca Current Modulation by a GABA Receptor J. Neurosci., May 1, 1998, 18(9):3138–3146 3139
Figure 1. Inhibitory effects of GABAB receptor agonists on EPSCs. EPSCs were evoked by extracellular stimulation. A (top row), Reversible inhibition of EPSCs by baclofen (2 ␮M) and attenuation of the baclofen effect by CGP35348 (100 ␮M) in an MNTB neuron. Bottom row, Inhibitory effect of GABA (20 ␮M) and its attenuation by CGP35348 in another MNTB neuron. The magnitude of inhibition by CGP35348 on the effect of baclofen and GABA was 82.8 Ϯ 2.3% (n ϭ 3) and 60.7 Ϯ 11% (n ϭ 3), respectively. B, Dose-dependent suppression of EPSCs by baclofen and GABA. Cumulative dose-dependent effects of baclofen (top) and GABA (bottom) on the amplitude of EPSCs recorded from MNTB neurons. Sample records from individual MNTB neurons are shown in the inset. Calibration: 2 nA, 10 msec. The curves fitted to data points derived from the following equation: magnitude of inhibition (%) ϭ maximal inhibition (%)/[1 ϩ (IC50/agonist concentration)n]. For baclofen and GABA, maximal inhibition was 82.0 and 90.8%, IC50 was 0.77 and 9.97 ␮M, and Hill coefficient (n) was 0.90 and 1.21, respectively. Magnitude of EPSC suppression by 20 ␮M baclofen was 80.1 Ϯ 2.6% at the cumulative-dose application (n ϭ 4), which was not significantly different ( p ϭ 0.13; Student’s t test) from that at the single-dose application both structures (Takahashi et al., 1996). EPSCs were evoked at 0.1 Hz by recording was typically 10 –20 M⍀ and was compensated by 60–90%.
extracellular stimulation of presynaptic axons near the midline of a slice Current or potential recordings were made with a patch-clamp amplifier with a bipolar platinum electrode (Barnes-Davies and Forsythe, 1995) in (Axopatch 200B, Axon Instruments, Foster City, CA). Unless noted a relatively thick slice (250 ␮m) or by presynaptic action potentials or otherwise, records were low-pass-filtered at 2.5–20 kHz and digitized at Ca 2ϩ currents elicited by a whole-cell pipette in thin slice (150 ␮m). The 5–50 kHz by a CED 1401 interface (Cambridge Electronic Design). Leak electrode resistances were 4 –7 M⍀ for the postsynaptic pipette and 6–10 currents were subtracted for presynaptic currents by a scaled pulse M⍀ for the presynaptic pipette. The series resistance of presynaptic divided by n (P/N) protocol. The liquid junctional potential between the 3140 J. Neurosci., May 1, 1998, 18(9):3138–3146
Takahashi et al. • Presynaptic Ca Current Modulation by a GABA Receptor Figure 2. Baclofen-induced suppression of EPSCs is blocked by GDP␤S. Simultaneous presynaptic and postsynaptic recordings at the calyx–MNTB synapse.
EPSCs were evoked by action potentials elicited by a depolarizing current pulse (2–10 msec) applied to a calyx through a whole-cell patch pipette. The postsyn- aptic holding potential was Ϫ70 mV. A, Reversible suppression of EPSCs by baclofen (20 ␮M). B, Block- ing effect of GDP␤S (tri-lithium salt, 3 mM) in the pipette on baclofen-induced suppression of EPSCs (a, b). A lower concentration of GDP␤S (0.2 mM) did not prevent the effect of baclofen (data not shown). After the pipettes were retracted, a second paired recording was made from the same structures with a presynaptic pipette containing GTP (0.5 mM) instead of GDP␤S.
Baclofen clearly suppressed the EPSCs (c, d), which gradually recovered after washout (e). Complete re- covery of EPSCs took 5–10 min (Fig. 7). When LiCl (9 mM) was included in the presynaptic pipette the ba- clofen effect was not attenuated (not shown). The amplitudes of EPSCs were normalized against the mean of the first seven (with GDP␤S) or six (with GTP) data points before baclofen application in each experiment; the data point represents means and the error bars represent SEMs derived from paired re- cording experiments at three different synapses. Ver- tical calibration scales indicate 80 mV for presynaptic membrane potentials (A and B) and 1.25 nA (A) or 0.6 nA ( B) for EPSCs. Scale bars, 10 msec.
pipette solution and aCSF was ϩ7.5 mV for solution A and ϩ3.3 mV for reported previously (Barnes-Davies and Forsythe, 1995), bath- solution B. The value of reversal potentials (see Fig. 6C) was corrected EPSCs in a reversible manner (Fig. 1A). This baclofen effect was switching superfusates by solenoid valves. Caged GTP␥S [S-(DMNPE- detectable at 0.2 ␮M, increased dose-dependently, and reached a caged) GTP␥S; Molecular Probes, Eugene, OR] was applied at 38 ␮M maximal at ϳ20 ␮M (Fig. 1B). Similarly, the inhibitory transmit- into calyces by dialysis from whole-cell pipettes. Care was taken to ter GABA suppressed EPSCs (Fig. 1). The 50% inhibitory con- protect the compound from short wavelength light during this procedure.
A flash of light was given from a mercury lamp light source (50 W) 50 ) of baclofen was estimated from the dose – through a filter (360 Ϯ 20 nm) by opening a shutter for a given period response curve to be 0.8 ␮M, whereas that for GABA was 10 ␮M (2– 4 sec). Application of the light flash without loading caged compound (Fig. 1B). Thus baclofen was about 10 times more potent than had no effect on the synaptic transmission or IpCa under normal exper- GABA in inhibiting EPSCs at this synapse. The inhibitory effects imental conditions, although an excessive illumination sometimes in- of both baclofen and GABA were largely attenuated by the duced a transient potentiation of IpCa or an increase in the frequency of spontaneous synaptic currents. Experiments were carried at room tem- GABAB receptor antagonist CGP35348 (100 ␮M) (Fig. 1A), in- dicating that the effects of baclofen and GABA were indeed Presynaptic inhibition mediated by GABA receptors at
Involvement of G-proteins in GABA receptor-
the calyx of Held
mediated presynaptic inhibition
A single extracellular stimulation evoked a large and rapidly An EPSC was evoked by a presynaptic action potential in a decaying EPSC in a principal cell of the medial nucleus of simultaneous whole-cell recording from the calyx of Held and a trapezoid body (MNTB) under whole-cell voltage clamp. As target MNTB cell (Fig. 2A). Baclofen suppressed the EPSC Takahashi et al. • Presynaptic Ca Current Modulation by a GABA Receptor J. Neurosci., May 1, 1998, 18(9):3138–3146 3141
are involved in the GABAB receptor-mediated presynaptic Inhibition of presynaptic calcium currents by baclofen
To identify an effector of the presynaptic GABAB receptor, we
first examined whether presynaptic calcium currents (IpCa) could be modulated by baclofen. As illustrated in Figure 3A, baclofen slowed activation kinetics of IpCa and reduced its amplitude.
When measured at the peak of the control current (1.3 msec from onset) at Ϫ10 mV, the magnitude of IpCa suppression was 38.0 Ϯ 3.8% (n ϭ 6). The baclofen-induced suppression of IpCa was not associated with a shift in the current–voltage (IV) relationship (Fig. 3C). As shown in Figure 3A,B, after a 10 msec depolarizing pulse (to Ϫ10 mV) IpCa deactivated exponentially with a fast time constant (0.14 Ϯ 0.03 msec; n ϭ 8). Baclofen had no effect on this deactivation time constant (0.14 Ϯ 0.05 msec after baclofen).
This suggests that baclofen has little effect on the presynaptic Ca2ϩ channel open time. These characteristics of the baclofen- induced inhibition of IpCa are similar to those reported for somatic Ca2ϩ currents (Dolphin and Scott, 1987; Scholtz and Miller, 1991; Mintz and Bean, 1993; Lambert and Wilson, 1996).
To study further the involvement of G-proteins in the baclofen- induced suppression of IpCa, caged GTP␥S (38 ␮M) was loaded into a calyx through a whole-cell patch pipette (Fig. 4A). After it was confirmed that baclofen reversibly suppressed IpCa (a–c), a flash of ultraviolet light (UV, 340–380 nm) was applied for 2–4 sec (arrow) to induce a photo-release of the caged GTP␥S com- pound. After the flash, IpCa gradually diminished in amplitude Figure 3. Suppression of presynaptic Ca 2ϩ currents by baclofen. The and slowed in its rising phase (c, d). After IpCa amplitude calyx was voltage-clamped at Ϫ80 mV, and IpCa was evoked by a 10 msec reached a steady level, a second application of baclofen no longer depolarizing pulse. In this experiment, [Ca 2ϩ]o was reduced to 1 mM attenuated IpCa (d, e). In agreement with this result, when to allow better voltage-clamp performance. A, IpCa induced in a calyx by a depolarizing voltage step to Ϫ10 mV in the absence and presence of ␥S (200 ␮M) was included in the presynaptic whole-cell baclofen (20 ␮M, superimposed). B, The tail currents are normalized at pipette, IpCa exhibited a similarly slow rise, and baclofen had no the peak and superimposed. C, Current–voltage relationships of IpCa significant effect on the current amplitude (99.8 Ϯ 1.6%; n ϭ 4) before (open circles) and after ( filled circles) baclofen application. Mean (Fig. 4B). When GDP␤S (3 mM) was included in the pipette, IpCa values Ϯ SEMs obtained from six calyces are shown.
had a normal rise time, but baclofen was again ineffective on IpCa (96.9 Ϯ 1.2%; n ϭ 5) (Fig. 4C). These results indicate that without affecting the presynaptic action potential. The magnitude the inhibitory effect of baclofen on the presynaptic calcium cur- of suppression of EPSCs by baclofen (20 ␮M) was 78.5 Ϯ 0.71% (mean Ϯ SEM; n ϭ 4 cells), which was comparable with that for the extracellularly evoked EPSCs (Fig. 1 and legend). The pre- Lack of baclofen effect on presynaptic
synaptic action potential had a peak amplitude of 90.5 Ϯ 12 mV potassium currents
and a half-width of 0.76 Ϯ 0.19 msec (n ϭ 4 calyces), which We next examined whether baclofen might modulate potassium remained at 99.0 Ϯ 2.4% and 113 Ϯ 25%, respectively, during currents. Voltage-dependent outward potassium currents were baclofen application. Baclofen had no effect on the presynaptic evoked by depolarizing a presynaptic terminal in the presence of membrane potential or conductance (see below).
TTX (1 ␮M) (Forsythe, 1994). As illustrated in Figure 5, the To directly address an involvement of presynaptic G-proteins potassium current before and after baclofen application was in the action of baclofen, the GDP analog guanosine-5Ј-O-(2- nearly identical at all voltages examined. Thus, GABAB receptors thiodiphosphate) (GDP␤S, 3 mM) was included in the presynaptic do not seem to be coupled with voltage-gated potassium channels whole-cell pipette. In this condition, baclofen no longer sup- pressed EPSCs (102 Ϯ 2.6%; n ϭ 4 pairs) (Fig. 2B). After the In neuronal somata, baclofen enhances inwardly rectifying whole-cell pipette containing GDP␤S was retracted, another potassium currents by activating G-proteins (Andrade et al., 1986; paired recording was made again at the same synapse, this time Sodickson and Bean, 1996). We examined whether baclofen with a presynaptic pipette solution containing GTP. Baclofen might similarly enhance the inward rectifying potassium current clearly suppressed EPSCs by 67 Ϯ 14% (n ϭ 3 pairs after GDP␤S at the presynaptic terminal. As illustrated in Figure 6A, baclofen washout) (Fig. 2B). Thus presynaptic GDP␤S blocked the effect applied at Ϫ70 mV holding potential had no effect on the holding of baclofen in a reversible manner. Similarly, when the nonhydro- current or the membrane conductance (98.5 Ϯ 1.8%; n ϭ 9) lyzable GTP analog guanosine 5Ј-O-(3-thiotriphosphate) measured by a ramp command voltage pulse (Fig. 6C). The (GTP␥S, 200 ␮M) was included in the presynaptic whole-cell inwardly rectifying potassium current is known to be blocked by pipette, baclofen had no effect on EPSCs (105 Ϯ 7.6%; n ϭ 3 a low concentration of Ba2ϩ (Hagiwara et al., 1978). Bath- pairs; data not shown). These results indicate that the effect of application of Ba2ϩ (100 ␮M) caused a small inward current baclofen on EPSCs is indeed presynaptic and that G-proteins accompanied by a slight decrease in membrane conductance (to 3142 J. Neurosci., May 1, 1998, 18(9):3138–3146
Takahashi et al. • Presynaptic Ca Current Modulation by a GABA Receptor Figure 4. Block of baclofen-induced IpCa sup- pression by GTP␥S or GDP␤S. A, Occlusion of baclofen effect by GTP␥S. IpCa was evoked in a calyx by a 20 msec depolarizing step from Ϫ70 mV to Ϫ13 mV. Baclofen (20 ␮M) suppressed IpCa, which recovered partially (a–c, superim- posed). After a light flash given at an arrow for 2 sec, IpCa diminished gradually (c, d). A second application of baclofen after the flash had no effect on IpCa (d, e). Essentially the same result was obtained in two other calyces. B, C, Little effect of baclofen on IpCa (evoked by a 20 msec depolarizing pulse from Ϫ80 mV to Ϫ10 mV) was observed in the presence of GTP␥S (200 ␮M, B) or GDP␤S (3 mM, C) in the presynaptic pi- pette. B and C are from different calyces. A similar result was obtained in another calyx for 84.5 Ϯ 4.6%; n ϭ 6) (Fig. 6A), suggesting that the inwardly rectifying channels might weakly contribute to the resting con-ductance of the presynaptic terminal. After a calyx was loadedwith caged GTP␥S, photo-release of GTP␥S by a flash (Fig. 6B, arrow) induced a prominent outward current accompanied by anincrease in membrane conductance. After the outward currentreached a steady level, subsequent application of Ba2ϩ (100 ␮M) largely abolished this current. When Ba2ϩ was washed out, theoutward current gradually recovered, with an increase in mem-brane conductance (not shown). The Ba2ϩ-sensitive current in-duced by GTP␥S was extracted as a difference current before and after the Ba2ϩ application (Fig. 6B, a and b). This currentrectified inwardly and reversed at Ϫ92 Ϯ 1.1 mV (n ϭ 4) close to the theoretical potassium equilibrium potential (99.5 mV; Fig. 6C,arrow), indicating that it is a G-protein-activated inwardly recti-fying potassium current (GIRK) (Kubo et al., 1993). Thus GIRKis present in the presynaptic terminal but cannot be activated byGABAB receptors.
Figure 5. Lack of baclofen effect on voltage-gated potassium currents.
Similar to Ba2ϩ, extracellular Csϩ blocks inwardly rectifying Inset, Outward potassium currents evoked by 20 mV depolarizing steps potassium currents (Hagiwara et al., 1976; Sodickson and Bean, from the holding potential of Ϫ80 mV to ϩ20 mV in the presence of TTX before (left) and after (middle) baclofen (20 ␮M) application. The super- 1996) as well as the inwardly rectifying cationic currents Ih imposed traces before and after baclofen application overlapped almost (Halliwell and Adams, 1982; Takahashi, 1990). Bath-application completely (right). The amplitude of the potassium current was normal- of Ba2ϩ or Csϩ (both at 1 mM) had no effect on EPSCs evoked ized against the value at 0 mV and mean Ϯ SEMs of five calyces before extracellularly (Fig. 7). Baclofen applied in the presence of Ba2ϩ (open circles) and after ( filled triangles) baclofen application are plotted or Csϩ suppressed EPSCs to a similar extent as in control: 72.5 Ϯ Takahashi et al. • Presynaptic Ca Current Modulation by a GABA Receptor J. Neurosci., May 1, 1998, 18(9):3138–3146 3143
Figure 6. Effects of baclofen and GTP␥S on presynaptic holding current and membrane conductance. A calyx was voltage-clamped at the holding potential of Ϫ70 mV, and a ramp command voltage from Ϫ50 to Ϫ130 mV (C, top left) was applied every 20 sec. A, Baclofen (20 ␮M) had no effect on the holding current or input conductance. Ba 2ϩ (100 ␮M) caused a slight inward current associated with a de- crease in conductance in the same calyx. B, In another calyx, photo-release of GTP␥S by a UV flash (arrow) induced an outward current accompanied by an increase in input conductance. This current was suppressed by Ba 2ϩ (100 ␮M, b). Application of the light flash without loading caged compound had no effect on the holding current or membrane conductance. The outward current was not ob- served after GTP␥S photolysis with the Csϩ-based internal solution for IpCa recordings (Fig. 4A). C, Currents (a, b, bottom) corresponding to a command voltage (top) after photolysis of caged GTP␥S compound before (a) and after ( b) application of Ba 2ϩ. Right, Ba 2ϩ-sensitive current ex- tracted as a difference current (a–b). Arrow indicates the- oretical equilibrium potential for potassium ions calculated from the internal and external potassium activities. The difference current between before and after photolysis had a similar reversal potential, but inward rectification was less prominent (data not shown). Membrane potential was cor- rected for the liquid junction potential between the exter- nal and internal solution (ϩ7.5 mV) for this current– voltage relationship. The data in this figure were low-pass- filtered at 100 Hz and sampled at 1 kHz.
4.3% in control (n ϭ 8), 72.6 Ϯ 2.0% in Ba2ϩ (n ϭ 4), and 74.8 Ϯ The result was essentially the same when the charge instead of the 1.5% in Csϩ (n ϭ 4), respectively. These results suggest further peak amplitude for IpCa and EPSCs was compared. Thus, the that neither GIRK nor Ih is involved in the GABAB receptor- baclofen-induced suppression of EPSCs can be explained mostly mediated presynaptic inhibition at the calyx–MNTB synapse.
Lack of contribution of exocytotic machinery to
GABA receptor-mediated presynaptic inhibition

DISCUSSION
To examine whether the exocytotic process downstream of Ca2ϩ Inhibition of calcium currents and transmission by
GABA receptor through G-protein
bition, we made simultaneous pre- and postsynaptic recordings In this study, using paired whole-cell recordings from the brain and compared the IpCa–EPSC relationship between two condi- stem giant presynaptic terminal and postsynaptic cell, we have tions: first after baclofen application and then after reduction of demonstrated that presynaptic GABAB receptors are linked [Ca2ϩ]o (Takahashi et al., 1996). When baclofen was applied, through G-proteins to Ca2ϩ channels, thereby suppressing trans- EPSCs diminished concomitantly with IpCa (Fig. 8A, i, ii). Sim- mitter release. The IpCa at the calyx–MNTB synapse is almost ilarly, when [Ca2ϩ]o was reduced by replacement with [Mg2ϩ]o, exclusively P-type at the age range examined (Forsythe et al., both EPSCs and IpCa were diminished in parallel (Fig. 8A, iii, 1998). It is possible that ␤␥ complex of heterotrimeric G-protein iv). When the IpCa–EPSC relations were plotted for data ob- may interact with the ␣1A subunit, thereby suppressing P-type tained after baclofen application and after [Ca2ϩ]o reduction, the Ca2ϩ channel activity (De Waard et al., 1997). Such a membrane- two relationships largely overlapped with each other (Fig. 8B). At delimited mechanism is consistent with our finding that the the seven synapses examined, the slope in the regression lines magnitude of the baclofen-induced suppression of EPSCs evoked after baclofen application was similar to that after [Ca2ϩ]o re- via a presynaptic whole-cell pipette was similar to that of EPSCs duction (Fig. 8B, inset) (no significant difference in paired t test).
evoked via an extracellular pipette. Thus diffusible intracellular 3144 J. Neurosci., May 1, 1998, 18(9):3138–3146
Takahashi et al. • Presynaptic Ca Current Modulation by a GABA Receptor ings from the calyx presynaptic terminals revealed that baclofen had no effect on the voltage-gated potassium currents or inwardly rectifying potassium currents. Furthermore, the inward rectifier channel blockers Ba2ϩ or Csϩ had no effect on the baclofen- induced inhibition of EPSCs. These results indicate that potas- sium conductances are not significantly involved in the GABAB receptor-mediated presynaptic inhibition at this fast excitatory In our present study, Ba2ϩ had no effect on GABAB receptor- mediated presynaptic inhibition as reported at other central syn- apses (Allerton et al., 1989; Lambert at al., 1991; Thompson and Gahwiler, 1992; Hirata et al., 1995). Although Ba2ϩ was reported to inhibit the effect of baclofen on monosynaptic IPSCs in hip- pocampal CA3 cells (Thompson and Gahwiler, 1992), this was not confirmed in a study using another blocking agent of inward rectifying potassium channels (Lambert and Wilson, 1993). Fur- thermore, transgenic mice lacking a GIRK gene exhibited a normal magnitude of GABAB receptor-mediated presynaptic in- hibition at hippocampal synapses (Luscher et al., 1997). Thus, so far there is no direct evidence to indicate an involvement of potassium conductances in the receptor-mediated presynaptic inhibition at mammalian central synapses.
At the calyx of Held, an inwardly rectifying potassium current could be activated by intracellular application of GTP␥S but not by baclofen. This result may imply that the GABAB receptors and G-proteins coupled with GIRK are distinct from those coupled with voltage-gated calcium channels, as proposed previously on the basis of pharmacological differences between the presynaptic and postsynaptic effect of baclofen (Dutar and Nicoll, 1988).
However, it is also possible that GIRK is localized outside of the functional domain of G-proteins coupled with GABAB receptors Figure 7. Effect of external Ba 2ϩ and Cs ϩ on GABAB receptor- mediated presynaptic inhibition. EPSCs were evoked in MNTB principal Baclofen had no effect on presynaptic spike waveform being cells by extracellular stimulation. A, Baclofen-induced suppression of consistent with the lack of involvement of potassium conductance EPSCs was similar before and after Ba 2ϩ application (1 mM). EPSCs before, during baclofen application (20 ␮M), and after washout are su- (also see Dittman and Regehr, 1996). Although baclofen sup- perimposed on top in the absence (left) and presence (right) of Ba 2ϩ.
pressed the presynaptic calcium conductance, this was not appar- Note the small polysynaptic EPSC component observed at the decay of ent in the action potential waveform. This might be attributable to monosynaptic EPSC. B, Baclofen suppressed EPSCs similarly in the the large potassium conductance masking the calcium conduc- absence and presence of external Cs ϩ (1 mM). EPSCs before and after tance. In fact, even after synaptic transmission was abolished by baclofen application are superimposed on top in the absence (left) and presence (right) of Cs ϩ. A and B are from different cells.
reducing [Ca2ϩ]o, the presynaptic action potential waveform remained similar at this (our unpublished observation) and other messengers, likely to be washed out during whole-cell recording, synapses (Sabatini and Regehr, 1997).
may not be essentially involved in the baclofen-induced suppres- sion of EPSCs. IpCa is also suppressed by a metabotropic gluta- The exocytotic machinery for evoked transmitter
release is not affected by GABA receptors

mate receptor (mGluR) agonist (Takahashi et al., 1996). It re- mains to be seen whether a common G-protein mediates the The IpCa–EPSC relationship during baclofen application was presynaptic inhibition by mGluRs and GABA similar to that during reduction of [Ca2ϩ]o. This suggests that the exocytotic machinery downstream of Ca2ϩ entry is not involved Presynaptic potassium channels are not coupled with
in GABAB receptor-mediated presynaptic inhibition at the calyx– GABA receptor
MNTB synapse, as is the case for mGluR-mediated presynaptic Presynaptic potassium conductances are thought to be important inhibition (Takahashi et al., 1996). In the case of baclofen, similar in the regulation of transmitter release (Augustine, 1990). The conclusions were made from studies using Ca2ϩ indicators at receptor-mediated inhibition of a potassium conductance is hippocampal synapses (Wu and Saggau, 1995; Dittman and known to enhance synaptic efficacy in invertebrate nervous sys- Regehr, 1997) (but see Dittman and Regehr, 1996). The direct tems (Kandel and Schwartz, 1982). At mammalian neuronal so- involvement of the exocytotic machinery in receptor-mediated mata, GABAB receptors potentiate transient potassium currents presynaptic inhibition has been postulated from the observation (Saint et al., 1990) or activate inwardly rectifying potassium that baclofen suppressed the frequency of spontaneous miniature currents through G-protein activation (Andrade et al., 1986; synaptic currents in a [Ca2ϩ]o-independent manner (Scanziani et Sodickson and Bean, 1996). It has been postulated that an en- al., 1992; Rohrbacher et al., 1997). However, differential modu- hancement of presynaptic potassium currents may underlie lations of the frequency of miniature events and the amplitude of GABAB receptor-mediated presynaptic inhibition (Saint et al., evoked synaptic responses by various manipulations are well 1990; Thompson and Gahwiler, 1992). However, direct record- known (Fu and Poo, 1991; Geppert et al., 1994; Cummings et al., Takahashi et al. • Presynaptic Ca Current Modulation by a GABA Receptor J. Neurosci., May 1, 1998, 18(9):3138–3146 3145
Figure 8. Comparison of IpCa–EPSC relationships during baclofen application and [Ca2ϩ]o reduction. Paired recording from a calyx and its target cell.
A, Effects of baclofen (20 ␮M; i, ii) and [Ca2ϩ]o reduction (iii, iv) on IpCa (Pre) and EPSCs (Post). IpCa was evoked by 1 msec depolarizing command pulse from Ϫ70 mV to Ϫ10 mV. Records before and after baclofen application or [Ca2ϩ]o reduction are superimposed on top row. B, Double logarithmic plot of IpCa–EPSC relation during baclofen application ( filled circles with a dotted regression line) and [Ca 2ϩ]o reduction (open circles with a solid regression line). Data points above 90% in EPSC amplitude were excluded from these plots to minimize constrainment. The slope value was 2.29 for baclofen and 2.33 for [Ca 2ϩ]o reduction, respectively. Excluding the minimal point from each relationship had no significant effect on the slope values (2.15 and 2.09, respectively, for baclofen and [Ca2ϩ]o reduction). Inset graph, The slope value of regression lines compared between [Ca2ϩ]o reduction and baclofen application at seven synapses. No significant difference with p ϭ 0.24 in paired t test. The mean slope value was 1.73 Ϯ 0.17 for baclofen and 1.77 Ϯ 0.17 for [Ca2ϩ]o reduction, respectively.
1996; Hori et al., 1996). A distinct mechanism may operate in the receptors at a giant excitatory synapse in rat auditory brainstem slices.
modulation of synchronous and asynchronous transmitter release.
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GABAB receptor-mediated presynaptic inhibition; neither potas- Cummings DD, Wilcox KS, Dichter MA (1996) Calcium-dependent sium channels nor modulation of the exocytotic machinery down- paired-pulse facilitation of miniature EPSC frequency accompanies stream of Ca2ϩ influx plays a significant role. On binding a ligand, depression of EPSCs at hippocampal synapses in culture. J Neurosci B receptors activate G-proteins and suppress De Waard M, Liu H, Walker D, Scott VES, Gurnett CA, Campbell KP Ca2ϩ currents, thereby reducing transmitter release. Given the (1997) Direct binding of G-protein ␤␥ complex to voltage-dependent wide distribution of presynaptic GABAB receptors at synapses calcium channels. Nature 385:446–450.
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