Presynaptic GABAB receptors play a regulatory role in central synaptic transmission. To elucidate their underlying mechanism of action, we have made whole-cell recordings of calcium and potassium currents from a giant presynaptic terminal, the calyx of Held, and EPSCs from its postsynaptic target in the medial nucleus of the trapezoid body of rat brainstem slices. The GABAB receptor agonist baclofen suppressed EPSCs and presynaptic calcium currents but had no effect on voltage-dependent potassium currents. The calcium current–EPSC relationship measured during baclofen application was similar to that observed on reducing [Ca2+]o, suggesting that the presynaptic inhibition generated by baclofen is caused largely by the suppression of presynaptic calcium influx. Presynaptic loading of the GDP analog guanosine-5′-O-(2-thiodiphosphate) (GDPβS) abolished the effect of baclofen on both presynaptic calcium currents and EPSCs. The nonhydrolyzable GTP analog guanosine 5′-O-(3-thiotriphosphate) (GTPγS) suppressed presynaptic calcium currents and occluded the effect of baclofen on presynaptic calcium currents and EPSCs. Photoactivation of GTPγS induced an inward rectifying potassium current at the calyx of Held, whereas baclofen had no such effect. We conclude that presynaptic GABAB receptors suppress transmitter release through G-protein-coupled inhibition of calcium currents.
- GABAB receptor
- presynaptic inhibition
- calcium currents
- inwardly rectifying potassium currents
- the calyx of Held
- presynaptic recording
GABAB receptors are widely distributed in the presynaptic and postsynaptic membranes of vertebrate central neurons, and they modulate synaptic transmission by either suppressing transmitter release or hyperpolarizing postsynaptic cells (Thompson et al., 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; Sodickson and Bean, 1996) or suppressing calcium channels (Dolphin and Scott, 1987; Scholtz and Miller, 1991; Mintz and Bean, 1993). Compared with the wealth of information on the postsynaptic mechanism of GABAB receptors, much less is known about their presynaptic mechanism of action. In particular, it is not known whether the effector of presynaptic GABAB receptors is a potassium channel (Saint et al., 1990; Thompson and Gahwiler, 1992), a calcium channel (Scholtz and Miller, 1991; Pfrieger et al., 1994; Wu and Saggau, 1995; Dittman and Regehr, 1996, 1997), or exocytotic machinery downstream of calcium influx (Scanziani et al., 1992; Dittman and Regehr, 1996; Rohrbacher et al., 1997). Also, an involvement of G-proteins in GABAB receptor-mediated presynaptic inhibition remains to be directly demonstrated (Thompson et al., 1993). The calyx of Held is an ideal preparation for directly testing these issues using patch-clamp techniques (Forsythe, 1994; Borst et al., 1995; Takahashi et al., 1996). The presynaptic terminal possesses GABAB receptors as well as metabotropic glutamate receptors and adenosine receptors (Barnes-Davies and Forsythe, 1995). Here we demonstrate that the G-protein-coupled inhibition of calcium channels underlies the GABAB receptor-mediated presynaptic inhibition.
MATERIALS AND METHODS
Preparation and solutions. Transverse slices of the superior olivary complex were prepared from 14- to 19-d-old Wistar rats killed by decapitation under halothane anesthesia. The medial nucleus of trapezoid body (MNTB) neurons and calyces were viewed with a 40×, 63× (Zeiss), or 60× (Olympus Optical, Tokyo, Japan) water immersion lens attached to an upright microscope (Axioskop, Zeiss). Each slice was perfused with artificial CSF (aCSF) containing 120 mmNaCl, 2.5 mm KCl, 26 mmNaHCO3, 1.25 mmNaH2PO4, 2 mmCaCl2, 1 mm MgCl2, 10 mm glucose, 0.5 mm myo-inositol, 2 mm sodium pyruvate, 0.5 mm ascorbic acid, and 4 mm lactic acid, pH 7.4, with 5% CO2 and 95% O2. To isolate Ca2+ currents, 10 mm tetraethylammonium (TEA) chloride and 1 μmtetrodotoxin (TTX) were included in the aCSF. The postsynaptic patch pipette was filled with a solution (A) containing 97.5 mmpotassium 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)-triethyl-ammonium bromide (QX314, 5 mm) was included in the postsynaptic pipette solution to suppress action potential generation when aCSF did not contain TTX. For recording EPSCs, the aCSF routinely contained bicuculline (10 μm) and strychnine (0.5 μm) to block spontaneous 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 mmMgCl2, 2 mm ATP, 0.5 mm GTP, 12 mm Na2 phosphocreatinine, and 10 mm TEA, pH adjusted to 7.4 with CsOH. Presynaptic potassium currents were recorded with solution A. The presynaptic pipette solutions routinely contained 2 mm ATP (ATP-Mg salt), 12 mm phosphocreatinine, and 0.5 mm GTP, unless noted otherwise. For paired recordings 10 mm potassium glutamate or cesium glutamate (equimolar replacement of KCl or CsCl) was also included in the presynaptic pipette solution (Borst et al., 1995; Takahashi et al., 1996).
Recording and data analysis. Whole-cell patch-clamp recordings were made from MNTB neurons, presynaptic calyces, or simultaneously from both structures (Takahashi et al., 1996). EPSCs were evoked at 0.1 Hz by extracellular stimulation of presynaptic axons near the midline of a slice with a bipolar platinum electrode (Barnes-Davies and Forsythe, 1995) in a relatively thick slice (250 μm) or by presynaptic action potentials or Ca2+currents elicited by a whole-cell pipette in thin slice (150 μm). The electrode resistances were 4–7 MΩ for the postsynaptic pipette and 6–10 MΩ for the presynaptic pipette. The series resistance of presynaptic recording was typically 10–20 MΩ and was compensated by 60–90%. Current or potential recordings were made with a patch-clamp amplifier (Axopatch 200B, Axon Instruments, Foster City, CA). Unless noted otherwise, records were low-pass-filtered at 2.5–20 kHz and digitized at 5–50 kHz by a CED 1401 interface (Cambridge Electronic Design). Leak currents were subtracted for presynaptic currents by a scaled pulse divided by n (P/N) protocol. The liquid junctional potential between the pipette solution and aCSF was +7.5 mV for solution A and +3.3 mV for solution B. The value of reversal potentials (see Fig. 6 C) was corrected for these junction potentials.
Drug application. GABAB receptor agonists were bath-applied by switching superfusates by solenoid valves. Caged GTPγS [S-(DMNPE-caged) GTPγS; Molecular Probes, Eugene, OR] was applied at 38 μm into calyces by dialysis from whole-cell pipettes. Care was taken to protect the compound from short wavelength light during this procedure. A flash of light was given from a mercury lamp light source (50 W) through a filter (360 ± 20 nm) by opening a shutter for a given period (2–4 sec). Application of the light flash without loading caged compound had no effect on the synaptic transmission or IpCa under normal experimental conditions, although an excessive illumination sometimes induced a transient potentiation of IpCa or an increase in the frequency of spontaneous synaptic currents. Experiments were carried at room temperature (22–26°C).
Presynaptic inhibition mediated by GABAB receptors at the calyx of Held
A single extracellular stimulation evoked a large and rapidly decaying EPSC in a principal cell of the medial nucleus of trapezoid body (MNTB) under whole-cell voltage clamp. As reported previously (Barnes-Davies and Forsythe, 1995), bath-application of the GABAB receptor agonist baclofen suppressed EPSCs in a reversible manner (Fig.1 A). This baclofen effect was detectable at 0.2 μm, increased dose-dependently, and reached a maximal at ∼20 μm (Fig.1 B). Similarly, the inhibitory transmitter GABA suppressed EPSCs (Fig. 1). The 50% inhibitory concentration (IC50) of baclofen was estimated from the dose–response curve to be 0.8 μm, whereas that for GABA was 10 μm (Fig. 1 B). Thus baclofen was about 10 times more potent than GABA in inhibiting EPSCs at this synapse. The inhibitory effects of both baclofen and GABA were largely attenuated by the GABAB receptor antagonist CGP35348 (100 μm) (Fig. 1 A), indicating that the effects of baclofen and GABA were indeed mediated by the GABAB receptor.
Involvement of G-proteins in GABAB receptor-mediated presynaptic inhibition
An EPSC was evoked by a presynaptic action potential in a simultaneous whole-cell recording from the calyx of Held and a target MNTB cell (Fig. 2 A). Baclofen suppressed the EPSC without affecting the presynaptic action potential. The magnitude of suppression of EPSCs by baclofen (20 μm) was 78.5 ± 0.71% (mean ± SEM;n = 4 cells), which was comparable with that for the extracellularly evoked EPSCs (Fig. 1 and legend). The presynaptic action potential had a peak amplitude of 90.5 ± 12 mV and a half-width of 0.76 ± 0.19 msec (n = 4 calyces), which remained at 99.0 ± 2.4% and 113 ± 25%, respectively, during baclofen application. Baclofen had no effect on the presynaptic membrane potential or conductance (see below).
To directly address an involvement of presynaptic G-proteins in the action of baclofen, the GDP analog guanosine-5′-O-(2-thiodiphosphate) (GDPβS, 3 mm) was included in the presynaptic whole-cell pipette. In this condition, baclofen no longer suppressed EPSCs (102 ± 2.6%;n = 4 pairs) (Fig. 2 B). After the whole-cell pipette containing GDPβS was retracted, another paired recording was made again at the same synapse, this time with a presynaptic pipette solution containing GTP. Baclofen clearly suppressed EPSCs by 67 ± 14% (n = 3 pairs after GDPβS washout) (Fig. 2 B). Thus presynaptic GDPβS blocked the effect of baclofen in a reversible manner. Similarly, when the nonhydrolyzable GTP analog guanosine 5′-O-(3-thiotriphosphate) (GTPγS, 200 μm) was included in the presynaptic whole-cell pipette, baclofen had no effect on EPSCs (105 ± 7.6%; n = 3 pairs; data not shown). These results indicate that the effect of baclofen on EPSCs is indeed presynaptic and that G-proteins are involved in the GABAB receptor-mediated presynaptic inhibition.
Inhibition of presynaptic calcium currents by baclofen
To identify an effector of the presynaptic GABABreceptor, we first examined whether presynaptic calcium currents (IpCa) could be modulated by baclofen. As illustrated in Figure3 A, 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 −10 mV, the magnitude of IpCa suppression was 38.0 ± 3.8% (n = 6). The baclofen-induced suppression of IpCa was not associated with a shift in the current–voltage (I–V) relationship (Fig. 3 C). As shown in Figure 3 A,B, after a 10 msec depolarizing pulse (to −10 mV) IpCa deactivated exponentially with a fast time constant (0.14 ± 0.03 msec;n = 8). Baclofen had no effect on this deactivation time constant (0.14 ± 0.05 msec after baclofen). This suggests that baclofen has little effect on the presynaptic Ca2+ channel open time. These characteristics of the baclofen-induced inhibition of IpCa are similar to those reported for somatic Ca2+ 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 GTPγS (38 μm) was loaded into a calyx through a whole-cell patch pipette (Fig.4 A). 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 GTPγS compound. After the flash, IpCa gradually diminished in amplitude and slowed in its rising phase (c, d). After IpCa amplitude reached a steady level, a second application of baclofen no longer attenuated IpCa (d, e). In agreement with this result, when GTPγS (200 μm) was included in the presynaptic whole-cell pipette, IpCa exhibited a similarly slow rise, and baclofen had no significant effect on the current amplitude (99.8 ± 1.6%;n = 4) (Fig. 4 B). When GDPβS (3 mm) was included in the pipette, IpCa had a normal rise time, but baclofen was again ineffective on IpCa (96.9 ± 1.2%;n = 5) (Fig. 4 C). These results indicate that the inhibitory effect of baclofen on the presynaptic calcium current is mediated by G-proteins.
Lack of baclofen effect on presynaptic potassium currents
We next examined whether baclofen might modulate potassium currents. Voltage-dependent outward potassium currents were evoked by depolarizing a presynaptic terminal in the presence of TTX (1 μm) (Forsythe, 1994). As illustrated in Figure5, the potassium current before and after baclofen application was nearly identical at all voltages examined. Thus, GABAB receptors do not seem to be coupled with voltage-gated potassium channels at the presynaptic terminal.
In neuronal somata, baclofen enhances inwardly rectifying potassium currents by activating G-proteins (Andrade et al., 1986; Sodickson and Bean, 1996). We examined whether baclofen might similarly enhance the inward rectifying potassium current at the presynaptic terminal. As illustrated in Figure6 A, baclofen applied at −70 mV holding potential had no effect on the holding current or the membrane conductance (98.5 ± 1.8%; n = 9) measured by a ramp command voltage pulse (Fig. 6 C). The inwardly rectifying potassium current is known to be blocked by a low concentration of Ba2+ (Hagiwara et al., 1978). Bath-application of Ba2+ (100 μm) caused a small inward current accompanied by a slight decrease in membrane conductance (to 84.5 ± 4.6%; n = 6) (Fig. 6 A), suggesting that the inwardly rectifying channels might weakly contribute to the resting conductance of the presynaptic terminal. After a calyx was loaded with caged GTPγS, photo-release of GTPγS by a flash (Fig. 6 B,arrow) induced a prominent outward current accompanied by an increase in membrane conductance. After the outward current reached a steady level, subsequent application of Ba2+ (100 μm) largely abolished this current. When Ba2+ was washed out, the outward current gradually recovered, with an increase in membrane conductance (not shown). The Ba2+-sensitive current induced by GTPγS was extracted as a difference current before and after the Ba2+ application (Fig. 6 B,a and b). This current rectified inwardly and reversed at −92 ± 1.1 mV (n = 4) close to the theoretical potassium equilibrium potential (99.5 mV; Fig.6 C, arrow), indicating that it is a G-protein-activated inwardly rectifying potassium current (GIRK) (Kubo et al., 1993). Thus GIRK is present in the presynaptic terminal but cannot be activated by GABAB receptors.
Similar to Ba2+, extracellular Cs+ blocks inwardly rectifying potassium currents (Hagiwara et al., 1976; Sodickson and Bean, 1996) as well as the inwardly rectifying cationic currents I h (Halliwell and Adams, 1982; Takahashi, 1990). Bath-application of Ba2+ or Cs+ (both at 1 mm) had no effect on EPSCs evoked extracellularly (Fig.7). Baclofen applied in the presence of Ba2+ or Cs+ suppressed EPSCs to a similar extent as in control: 72.5 ± 4.3% in control (n = 8), 72.6 ± 2.0% in Ba2+(n = 4), and 74.8 ± 1.5% in Cs+ (n = 4), respectively. These results suggest further that neither GIRK nor I his involved in the GABAB receptor-mediated presynaptic inhibition at the calyx–MNTB synapse.
Lack of contribution of exocytotic machinery to GABABreceptor-mediated presynaptic inhibition
To examine whether the exocytotic process downstream of Ca2+ influx is involved in GABABreceptor-mediated presynaptic inhibition, we made simultaneous pre- and postsynaptic recordings and compared the IpCa–EPSC relationship between two conditions: first after baclofen application and then after reduction of [Ca2+]o (Takahashi et al., 1996). When baclofen was applied, EPSCs diminished concomitantly with IpCa (Fig. 8 A,i, ii). Similarly, when [Ca2+]o was reduced by replacement with [Mg2+]o, both EPSCs and IpCa were diminished in parallel (Fig. 8 A,iii, iv). When the IpCa–EPSC relations were plotted for data obtained after baclofen application and after [Ca2+]o reduction, the two relationships largely overlapped with each other (Fig.8 B). At the seven synapses examined, the slope in the regression lines after baclofen application was similar to that after [Ca2+]o reduction (Fig.8 B, inset) (no significant difference in paired t test). The result was essentially the same when the charge instead of the peak amplitude for IpCa and EPSCs was compared. Thus, the baclofen-induced suppression of EPSCs can be explained mostly by a reduction of IpCa.
Inhibition of calcium currents and transmission by GABAB receptor through G-protein
In this study, using paired whole-cell recordings from the brain stem giant presynaptic terminal and postsynaptic cell, we have demonstrated that presynaptic GABAB receptors are linked through G-proteins to Ca2+ channels, thereby suppressing transmitter release. The IpCa at the calyx–MNTB synapse is almost exclusively P-type at the age range examined (Forsythe et al., 1998). It is possible that βγ complex of heterotrimeric G-protein may interact with the α1A subunit, thereby suppressing P-type Ca2+ channel activity (De Waard et al., 1997). Such a membrane-delimited mechanism is consistent with our finding that the magnitude of the baclofen-induced suppression of EPSCs evoked via a presynaptic whole-cell pipette was similar to that of EPSCs evoked via an extracellular pipette. Thus diffusible intracellular messengers, likely to be washed out during whole-cell recording, may not be essentially involved in the baclofen-induced suppression of EPSCs. IpCa is also suppressed by a metabotropic glutamate receptor (mGluR) agonist (Takahashi et al., 1996). It remains to be seen whether a common G-protein mediates the presynaptic inhibition by mGluRs and GABAB receptors.
Presynaptic potassium channels are not coupled with GABAB receptor
Presynaptic potassium conductances are thought to be important in the regulation of transmitter release (Augustine, 1990). The receptor-mediated inhibition of a potassium conductance is known to enhance synaptic efficacy in invertebrate nervous systems (Kandel and Schwartz, 1982). At mammalian neuronal somata, GABABreceptors potentiate transient potassium currents (Saint et al., 1990) or activate inwardly rectifying potassium currents through G-protein activation (Andrade et al., 1986; Sodickson and Bean, 1996). It has been postulated that an enhancement of presynaptic potassium currents may underlie GABAB receptor-mediated presynaptic inhibition (Saint et al., 1990; Thompson and Gahwiler, 1992). However, direct recordings 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 Ba2+ or Cs+ had no effect on the baclofen-induced inhibition of EPSCs. These results indicate that potassium conductances are not significantly involved in the GABAB receptor-mediated presynaptic inhibition at this fast excitatory synapse.
In our present study, Ba2+ had no effect on GABAB receptor-mediated presynaptic inhibition as reported at other central synapses (Allerton et al., 1989; Lambert at al., 1991;Thompson and Gahwiler, 1992; Hirata et al., 1995). Although Ba2+ was reported to inhibit the effect of baclofen on monosynaptic IPSCs in hippocampal 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). Furthermore, transgenic mice lacking a GIRK gene exhibited a normal magnitude of GABAB receptor-mediated presynaptic inhibition 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 GTPγS 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 at the presynaptic nerve terminal.
Baclofen had no effect on presynaptic spike waveform being consistent with the lack of involvement of potassium conductance (also see Dittman and Regehr, 1996). Although baclofen suppressed the presynaptic calcium conductance, this was not apparent in the action potential waveform. This might be attributable to the large potassium conductance masking the calcium conductance. In fact, even after synaptic transmission was abolished by reducing [Ca2+]o, the presynaptic action potential waveform remained similar at this (our unpublished observation) and other synapses (Sabatini and Regehr, 1997).
The exocytotic machinery for evoked transmitter release is not affected by GABAB receptors
The IpCa–EPSC relationship during baclofen application was similar to that during reduction of [Ca2+]o. This suggests that the exocytotic machinery downstream of Ca2+ entry is not involved in GABAB receptor-mediated presynaptic inhibition at the calyx–MNTB synapse, as is the case for mGluR-mediated presynaptic inhibition (Takahashi et al., 1996). In the case of baclofen, similar conclusions were made from studies using Ca2+ indicators at hippocampal synapses (Wu and Saggau, 1995; Dittman and Regehr, 1997) (but see Dittman and Regehr, 1996). The direct involvement of the exocytotic machinery in receptor-mediated presynaptic inhibition has been postulated from the observation that baclofen suppressed the frequency of spontaneous miniature synaptic currents in a [Ca2+]o-independent manner (Scanziani et al., 1992; Rohrbacher et al., 1997). However, differential modulations of the frequency of miniature events and the amplitude of evoked synaptic responses by various manipulations are well known (Fu and Poo, 1991; Geppert et al., 1994; Cummings et al., 1996; Hori et al., 1996). A distinct mechanism may operate in the modulation of synchronous and asynchronous transmitter release.
In conclusion, this study has demonstrated G-protein-coupled modulation of presynaptic Ca2+ channels on activation of GABAB receptor-mediated presynaptic inhibition; neither potassium channels nor modulation of the exocytotic machinery downstream of Ca2+ influx plays a significant role. On binding a ligand, presynaptic GABAB receptors activate G-proteins and suppress Ca2+ currents, thereby reducing transmitter release. Given the wide distribution of presynaptic GABAB receptors at synapses throughout the nervous system, this mechanism would be of general application.
This work was supported by the “Research for the Future” Program by The Japan Society for the Promotion of Sciences. We thank M. Farrant, I. D. Forsythe, T. Manabe, and K. Kobayashi for critically reading this manuscript. We are also 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 generous gift of CGP35348.
Correspondence should be addressed to Tomoyuki Takahashi, Department of Neurophysiology, University of Tokyo Faculty of Medicine, Tokyo 113, Japan.