The Journal of Neurophysiology Vol. 80 No. 3 September 1998, pp. 1571-1576
Copyright ©1998 by the American Physiological Society

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GABA_B Receptor-Mediated Modulation of Presynaptic Currents and Excitatory Transmission at a Fast Central Synapse

Jeffry S. Isaacson

Department of Physiology and Biophysics, University of Washington, Seattle, Washington 98195

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Isaacson, Jeffry S. GABA_B receptor-mediated modulation of presynaptic currents and excitatory transmission at a fast central synapse. J. Neurophysiol. 80: 1571-1576, 1998. Large nerve terminals (calyces of Held) in the medial nucleus of the trapezoid body (MNTB) offer a unique opportunity to explore the modulation of presynaptic channels at a mammalian central synapse. In this study I examined -aminobutyric acid-B (GABA_B)-mediated presynaptic inhibition at the calyx of Held in slices of the rat auditory brain stem. The selective GABA_B agonist baclofen caused a potent inhibition of synaptic transmission and presynaptic Ca²⁺ current. The inhibition of presynaptic Ca²⁺ channels was associated with a slowing of the activation kinetics of the underlying current, and the inhibition was relieved by strong depolarization. The inhibition of both synaptic transmission and presynaptic Ca²⁺ current was abolished by N-ethylmaleimide, a sulfhydryl alkylating agent that uncouples the G_o/G_i class of G proteins from receptors. Baclofen does not activate a potassium conductance in the presynaptic terminal. Taken together, these results suggest that GABA_B receptors inhibit synaptic transmission via G protein-mediated modulation of presynaptic Ca²⁺ channels at this large central synapse. Furthermore, these findings demonstrate that basic mechanisms of G protein-mediated inhibition of Ca²⁺ channels, proposed from recordings of neuron cell bodies, are well conserved at nerve endings in the mammalian brain.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

At many central synapses, -aminobutyic acid-B (GABA_B) receptors modulate transmission by inhibitory effects on nerve terminals (Nicoll et al. 1990). Studies of somatic currents found that GABA_B receptors are coupled via G proteins to the inhibition of Ca²⁺ channels (Dolphin and Scott 1987), and inhibition of presynaptic Ca²⁺ channels was suggested to mediate the action of GABA_B receptors on synaptic transmission (Dittman and Regehr 1996; Wu and Saggau 1997). However, GABA_B receptors hyperpolarize neurons through the activation of G protein-coupled inwardly rectifying potassium channels (GIRKs) (Andrade et al. 1986; Sodickson and Bean 1996), and it was reported that the K⁺ channel blocker Ba²⁺ can reduce GABA_B-mediated presynaptic inhibition (Misgeld et al. 1989; Thompson and Gähwiler 1992). Therefore the mechanism of action of GABA_B receptors on channels in nerve terminals is unclear.

Studies of the giant synapse of the avian ciliary ganglion have described the modulation of presynaptic Ca²⁺ channels (Bennett and Ho 1991; Stanley and Mirotznick 1997; Yawo and Chuhma 1993). However, the small size of most synaptic structures in the mammalian brain made direct electrophysiological studies of presynaptic terminals and their modulation impossible. In mammals, single nerve fibers originating from bushy cells of the ventral cochlear nucleus make unusually large contacts (the calyces of Held) onto the cell bodies of principal cells in the medial nucleus of the trapezoid body (MNTB) (Barnes-Davies and Forsythe 1995; Borst and Sakmann 1996; Takahashi et al. 1996). In this study, I used patch-clamp techniques to examine directly the actions of GABA_B receptors on presynaptic channels in calyx nerve terminals of the mammalian auditory brain stem.

	METHODS

Abstract Introduction Methods Results Discussion References

Transverse slices (150-200 µm) of the brain stem containing the MNTB were prepared from 8- to 17-day-old Sprague-Dawley rats with the use of conventional methods (Isaacson and Walmsley 1995) and visualized with IR-DIC optics (Zeiss Axioskop). Slices were perfused with a Ringer solution containing (in mM) 119 NaCl, 2.5 KCl, 1.3 Mg₂SO₄, 2.5 CaCl₂, 11 glucose, and 0.01 strychnine equilibrated with 95% O₂-5% CO₂. Patch pipettes (1.5-5 M) contained (in mM) 110 CsCl, 20 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 12 phosphocreatine, 10 tetraethylammonium chloride (TEACl), 3 MgATP, 0.5 sodium guanosine 5'-triphosphate (NaGTP), and 0.2 or 10 ethylene glycol-bis(-aminoethyl ether)-N,N,N,'N,'-tetraacetic acid (pH 7.3). The internal solution was supplemented with Lucifer yellow (0.3 mg/ml; K⁺ salt), and presynaptic recordings were confirmed at the end of each experiment with the use of epifluorescent illumination. Series resistance (<8 M in postsynaptic recordings and <20 M in presynaptic recordings) was routinely compensated by >80%. Synaptic currents were evoked via a Ringer-filled pipette (10-µm tip) placed at the midline. Presynaptic Ca²⁺ currents were isolated in the presence of tetrodotoxin (TTX, 1 µM), TEA (5 mM), and 4-aminopyridine (100 µM). Leak currents were subtracted on-line with a P/4 protocol. Ca²⁺ current amplitude was measured as the average value between 2 and 3 ms after the start of the voltage step. The activation time course of the Ca²⁺ current was determined by fitting a single exponential to the first 5 ms of the current traces (_act). Prepulse facilitation was studied by using pairs of pulses (1-s interval) delivered every 10 s. The second pulse began 20 ms after a preceding 40-ms step to +120 mV. In experiments studying presynaptic K⁺ currents, the superfusing solution was supplemented with TTX (1 µM), and KMeSO₄ replaced the Cs- and TEACl in the internal solution. Currents were recorded and filtered at 2-5 kHz with an Axopatch 1D amplifier (Axon Instruments) before being digitized at 4-10 kHz. Action potentials were recorded with the same amplifier in current-clamp mode. Data were analyzed with the use of pClamp 6 (Axon Instruments). Unless stated otherwise, postsynaptic currents and presynaptic currents were recorded at holding potentials of -30 and -80 mV, respectively. All experiments were performed at room temperature (22-25°C). Results are shown as means ± SE. Significance was determined with the use of a paired t-test.

	RESULTS

Abstract Introduction Methods Results Discussion References

At a holding potential of -70 mV, stimulation at the midline of the brain stem evoked large (>2 nA peak amplitude) and fast (decay time constant ~1 ms) excitatory postsynaptic currents (EPSCs) in principal cells of the MNTB (Fig. 1A₁). These responses were mediated by single nerve fibers because they occurred in an all-or-none fashion as the stimulus strength was increased (not shown). Membrane depolarization revealed a slowly decaying component of the EPSC that was most evident beyond the reversal potential (~0 mV) of the response (Fig. 1A₁). The slow component was blocked by D-amino-5-phosphonovaleric acid (D-APV; 25 µM), and the fast component was abolished by 6,7-dinitro-quinoxaline-2,3-dione (DNQX; 10 µM, n = 3, Fig. 1A₂). These results confirm that the calyx EPSC is mediated by both N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors (Barnes-Davies and Forsythe 1995).

View larger version (25K): [in this window] [in a new window]   FIG. 1. Calyx excitatory postsynaptic currents (EPSCs) are blocked by baclofen. A1: stimulation at the midline evokes large amplitude dual-component EPSCs in principal cells of the medial nucleus of the trapezoid body (MNTB). EPSCs were evoked at holding potentials shown to the left of each trace, and the inset shows the I-V relationship of the fast () and slow component (). A2: slow component of the EPSC is blocked by amino-5-phosphonovaleric acid (APV; 25 µM), and the remaining fast component is blocked by 6,7-dinitro-quinoxaline-2,3-dione (DNQX; 10 µM). B: baclofen (Bac, 50 µM) inhibits the dual-component EPSC. Traces are from the experiment plotted below.

Baclofen (50 µM) caused a rapid reduction in the amplitude of the evoked EPSC (Fig. 1B), in agreement with recent findings of GABA_B inhibition at the calyx synapse (Barnes-Davies and Forsythe 1995). The peak amplitude of the non-NMDA component was reduced by 88 ± 2% (SE, n = 6). Whole terminal voltage-clamp recordings were next used to determine the presynaptic action of baclofen (Fig. 2, A and B). Voltage steps from -80 mV evoked inward currents that peaked at approximately -10 mV (1.9 ± 0.2 nA, n = 11) and appeared to reverse at a potential of approximately +60 mV (Fig. 2C). Cadmium (100 µM) completely blocked these inward currents, indicating that they were Ca²⁺ channel mediated (Fig. 2D, n = 6). Baclofen caused a rapid reduction (46 ± 3%, Fig. 3, A and B) in the amplitude of nerve terminal Ca²⁺ currents in all calyces examined (n = 11). The inhibition was evident as a marked slowing of the activation kinetics of the current during a voltage step to 0 mV (control _act = 0.40 ± 0.03 ms, baclofen _act = 0.81 ± 0.14 ms, n = 8, P < 0.02). These results show that baclofen potently inhibits both the EPSC and nerve terminal Ca²⁺ channels.

View larger version (74K): [in this window] [in a new window]   FIG. 2. Voltage-clamp recordings of nerve terminal Ca2+ currents A: IR-DIC video image of a principal cell and calyx. B: image of the same field under fluorescent illumination. The terminal was filled with Lucifer yellow via a patch electrode. Thin process is the axon. Scale bar is 5 µm in A and B. C: I-V relationship of currents evoked with the use of depolarizing steps from a holding potential of -80 mV. Amplitudes represent the early component of the current. D: Ca2+ currents are abolished by cadmium (100 µM).

View larger version (21K): [in this window] [in a new window]   FIG. 3. Baclofen causes voltage-dependent inhibition of presynaptic Ca2+ channels. A1: baclofen (50 µM) reversibly reduces Ca2+ currents in the nerve terminal. A2: time course of the baclofen-induced inhibition from another calyx. B1: strong depolarization relieves -aminobutyric acid-B (GABAB) receptor-mediated inhibition of nerve terminal Ca2+ currents. Ca2+ currents were evoked with and without a prepulse to +120 (protocol not drawn to scale). B2: bar graph summarizes the effects of baclofen on Ca2+ current of control responses and those preceded by strong depolarization (n = 5).

Recordings from isolated cell bodies established that a major signaling pathway mediating the inhibition of Ca²⁺ channels by G protein-coupled receptors can be relieved by strong depolarization (Bean 1989; Hille 1994). A paired pulse protocol (Shapiro et al. 1994) was next used to look for a voltage dependence of GABA_B-mediated inhibition in the calyx nerve terminal (Fig. 3B₁). A prepulse to +120 mV reduced markedly (P < 0.01) the inhibition in response to baclofen (50 ± 6% vs. 34 ± 7% inhibition of the control and conditioned response, respectively, n = 5).

N-ethylmaleimide (NEM), a sulfhydryl alkylating agent, selectively uncouples pertussis toxin-sensitive G proteins from receptors (Jakobs et al. 1982; Shapiro et al. 1994). I next studied the action of NEM (50 µM) on the EPSC and presynaptic Ca²⁺ current. NEM caused a small and variable increase(36 ± 20%, n = 4) in the amplitude of the non-NMDA component of the EPSC. In the presence of NEM (5-10 min), baclofen caused only a weak inhibition of the EPSC (7 ± 2%, n = 6, Fig. 4A). Figure 4B illustrates the actions of NEM on presynaptic Ca²⁺ current in a representative recording. Baclofen caused a marked inhibition of the Ca²⁺ current under control conditions, but this effect was virtually abolished after NEM was added to the superfusing solution. This was not caused by a loss of responsiveness to agonist because baclofen caused robust inhibition when applied repeatedly to control slices (not shown). NEM caused a small increase in the amplitude of presynaptic Ca²⁺ currents (11 ± 6%, n = 4). In the presence of NEM, baclofen produced only a slight reduction of presynaptic Ca²⁺ currents (6 ± 1%). The actions of baclofen under control conditions and in the presence of NEM are summarized in Fig. 4C.

View larger version (25K): [in this window] [in a new window]   FIG. 4. GABAB-mediated inhibition of the EPSC, and presynaptic Ca2+ channels is N-ethylmaleimide (NEM) sensitive. A: NEM (50 µM) blocks the ability of baclofen (Bac, 50 µM) to inhibit the EPSC. Sweeps represent time points indicated in the graph. B: NEM (50 µM) blocks the ability of baclofen (Bac, 50 µM) to inhibit presynaptic Ca2+ current. Voltage steps were evoked to 0 mV from a holding potential of -80 mV. C: summary of baclofen-induced inhibition of the EPSC and presynaptic Ca2+ current (ICa) under control conditions and in the presence of NEM. Numbers in parentheses are the number of observations. D: baclofen (50 µM) has no effect on currents elicited by a voltage ramp with the use of a K+-based internal solution. Inset: baclofen does not alter the action potential properties of a terminal recorded in current clamp. Vrest, -75 mV; current step, 100 pA.

Although GABA_B receptors activate GIRKs in the dendrites and cell bodies of central neurons (Nicoll et al. 1990; Sodickson and Bean 1996), it is unclear if GIRKs are coupled to GABA_B receptors in nerve terminals. I next tested for the presence of baclofen-induced K⁺ currents with the use of a voltage-ramp protocol (-170 to -40 mV, 50-ms duration). Baclofen did not induce a current over this voltage range (Fig. 4D) nor did it cause a change in the properties of presynaptic action potentials recorded in current clamp (Fig. 4D, inset) in all cells examined (n = 4). These results indicate that GABA_B receptors do not activate K⁺ channels in the calyx nerve terminal.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

Whereas many G protein-coupled receptors were shown to inhibit Ca²⁺ currents in the cell bodies of mammalian central neurons (Hille 1994; Nicoll et al. 1990), their actions at nerve endings are less clear. At the calyx nerve terminal, baclofen caused a rapid inhibition of presynaptic Ca²⁺ current. This was associated with a slowing in the activation kinetics of the Ca²⁺ channels. Furthermore, strong depolarization relieved GABA_B-mediated inhibition. These actions are hallmarks of membrane-delimited, G protein-mediated inhibition (Hille 1994) in which channels undergo a shift in their voltage dependence of gating (Bean 1989). The G proteins involved in the inhibition of synaptic transmission and presynaptic Ca²⁺ channels are presumably of the G_o/G_i class because NEM abolished the actions of baclofen. The small facilitating action of NEM by itself suggests a relief of tonic inhibition that may reflect agonist-independent G protein activation or, alternatively, the activation of receptors by ambient levels of neuromodulators in the slice.

Although GABA_B receptors mediate postsynaptic inhibition via the activation of GIRKs in central neurons (Nicoll et al. 1990), baclofen did not induce a K⁺ conductance in the calyx nerve terminal. These results are consistent with a recent study of transgenic mice lacking the GIRK2 gene (Lüscher et al. 1997) and suggest that the activation of presynaptic K⁺ channels does not contribute to GABA_B-mediated inhibition of synaptic transmission.

Given that transmitter release at the calyx synapse has a highly nonlinear dependence on presynaptic Ca²⁺ influx (Borst and Sakmann 1996; Takahashi et al. 1996), the 50% reduction in presynaptic Ca²⁺ current is likely to account for the large (90%) reduction in EPSC amplitude. Although these findings do not rule out a direct effect on the release machinery itself (Dittman and Regehr 1996; Scanziani et al. 1992), these results are consistent with Ca²⁺ channels playing a major role in presynaptic inhibition (Dittman and Regehr 1996; Dunlap and Fischbach 1981; Takahashi et al. 1996; Wu and Saggau 1997). Furthermore, these findings indicate that the basic mechanisms of G protein-mediated inhibition of Ca²⁺ channels proposed from somatic recordings are well conserved at nerve endings in the mammalian brain.

	ACKNOWLEDGEMENTS

  I am indebted to Drs. Albert Berger, in whose lab this work was performed, and Bertil Hille for support and helpful advice. I thank Dr. Mark Shapiro for comments on the manuscript.

  This work was supported by a Burroughs Wellcome Fund Career Award in Biomedical Science.

	FOOTNOTES

  Address for reprint requests: Dept. of Physiology and Biophysics, Box 357290, University of Washington, Seattle, WA 98195.

  Received 3 April 1998; accepted in final form 8 June 1998.

	REFERENCES

Abstract Introduction Methods Results Discussion References

• ANDRADE, R., MALENKA, R. C., NICOLL, R. A. A G protein couples serotonin and GABA_B receptors to the same channels in hippocampus. Science 234: 1261-1265, 1986.[Abstract/Free Full Text]
• BARNES-DAVIES, M., FORSYTHE, I. D. Pre- and postsynaptic glutamate receptors at a giant excitatory synapse in rat auditory brainstem slices. J. Physiol. (Lond.) 488: 387-406, 1995.[Abstract/Free Full Text]
• BEAN, B. P. Neurotransmitter inhibition of neuronal calcium currents by changes in channel voltage dependence. Nature 340: 153-156, 1989.[Medline]
• BENNETT, M. R., HO, S. Probabilistic secretion of quanta from nerve terminals in avian ciliary ganglia modulated by adenosine. J. Physiol. (Lond.) 440: 513-527, 1991.[Abstract/Free Full Text]
• BORST, J. G., SAKMANN, B. Calcium influx and transmitter release in a fast CNS synapse. Nature 383: 431-434, 1996.[Medline]
• DITTMAN, J. S., REGEHR, W. G. Contributions of calcium-dependent and calcium-independent mechanisms to presynaptic inhibition at a cerebellar synapse. J. Neurosci. 16: 1623-1633, 1996.[Abstract/Free Full Text]
• DOLPHIN, A. C., SCOTT, R. H. Calcium channel currents and their modulation by (-)-baclofen in rat sensory neurones: modulation by guanine nucleotides. J. Physiol. (Lond.) 386: 1-17, 1987.[Abstract/Free Full Text]
• DUNLAP, K., FISCHBACH, G. D. Neurotransmitters decrease the calcium conductance activated by depolarization of embryonic chick sensory neurones. J. Physiol. (Lond.) 317: 519-535, 1981.[Abstract/Free Full Text]
• HILLE, B. Modulation of ion-channel function by G-protein-coupled receptors. Trends Neurosci. 17: 531-536, 1994.[Medline]
• ISAACSON, J. S., WALMSLEY, B. Receptors underlying excitatory synaptic transmission in slices of the rat anteroventral cochlear nucleus. J. Neurophysiol. 73: 964-973, 1995.[Abstract/Free Full Text]
• JAKOBS, K. H., LASCH, P., MINUTH, M., AKTORIES, K., SCHULTZ, G. Uncoupling of -adrenoreceptor-mediated inhibition of human platelet adenylate cyclase by N-ethylmaleimide. J. Biol. Chem. 257: 2829-2833, 1982.[Abstract/Free Full Text]
• LÜSCHER, C., JAN, L. Y., STOFFEL, M., MALENKA, R. C., NICOLL, R. A. G protein-coupled inwardly rectifying K⁺ channels (GIRKs) mediate postsynaptic but not presynaptic transmitter actions in hippocampal neurons. Neuron. 19: 687-695, 1997.[Medline]
• MISGELD, U., MULLER, W., BRUNNER, H. Effects of (-)baclofen on inhibitory neurons in the guinea pig hippocampal slice. Pflügers Arch. 414: 139-144, 1989.[Medline]
• NICOLL, R. A., MALENKA, R. C., KAUER, J. A. Functional comparison of neurotransmitter receptor subtypes in mammalian central nervous system. Physiol. Rev. 70: 513-565, 1990.[Free Full Text]
• SCANZIANI, M., CAPOGNA, M., GÄHWILER, B. H., THOMPSON, S. M. Presynaptic inhibition of miniature excitatory synaptic currents by baclofen and adenosine in the hippocampus. Neuron 9: 919-927, 1992.[Medline]
• SHAPIRO, M. S., WOLLMUTH, L. P., HILLE, B. Modulation of Ca²⁺ channels by PTX-sensitive G-proteins is blocked by N-ethylmaleimide in rat sympathetic neurons. J. Neurosci. 14: 7109-7116, 1994.[Abstract]
• SODICKSON, D. L., BEAN, B. P. GABA_B receptor-activated inwardly rectifying potassium current in dissociated hippocampal CA3 neurons. J. Neurosci. 16: 6374-6385, 1996.[Abstract/Free Full Text]
• STANLEY, E. F, MIROTZNICK, R. R. Cleavage of syntaxin prevents G-protein regulation of presynaptic calcium channels. Nature 385: 340-343, 1997.[Medline]
• TAKAHASHI, T., FORSYTHE, I. D., TSUJIMOTO, T., BARNES-DAVIES, M., ONODERA, K. Presynaptic calcium current modulation by a metabotropic glutamate receptor. Science 274: 594-597, 1996.[Abstract/Free Full Text]
• THOMPSON, S. M., GÄHWILER, B. H. Comparison of the actions of baclofen at pre- and postsynaptic receptors in the rat hippocampus in vitro. J. Physiol. (Lond.) 451: 329-345, 1992.[Abstract/Free Full Text]
• WU, L. G., SAGGAU, P. Presynaptic inhibition of elicited neurotransmitter release. Trends Neurosci. 20: 204-212, 1997.[Medline]
• YAWO, H., CHUHMA, N. Preferential inhibition of omega-conotoxin-sensitive presynaptic Ca²⁺ channels by adenosine autoreceptors. Nature 365: 256-258, 1993.[Medline]

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