G protein subunits mediate presynaptic inhibition of transmitter release from rat superior cervical ganglion neurones in culture
1 Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, UK
2 Department of Physiology, Tokyo Medical University, Tokyo 160-8402, Japan
Abstract
The activation of presynaptic G protein-coupled receptors (GPCRs) is widely reported to inhibit transmitter release; however, the lack of accessibility of many presynaptic terminals has limited direct analysis of signalling mediators. We studied GPCR-mediated inhibition of fast cholinergic transmission between superior cervical ganglion neurones (SCGNs) in culture. The adrenoceptor agonist noradrenaline (NA) caused a dose-related reduction in evoked excitatory postsynaptic potentials (EPSPs). NA-induced EPSP decrease was accompanied by effects on the presynaptic action potential (AP), reducing AP duration and amplitude of the after-hyperpolarization (AHP), without affecting the pre- and postsynaptic membrane potential. All effects of NA were blocked by yohimbine and synaptic transmission was reduced by clonidine, consistent with an action at presynaptic 2-adrenoceptors. NA-induced inhibition of transmission was sensitive to pre-incubation of SCGNs with pertussis toxin (PTX), implicating the involvement of Gi/o subunits. Expression of G transducin, an agent which sequesters G protein (G) subunits, in the presynaptic neurone caused a time-dependent attenuation of NA-induced inhibition. Injection of purified G subunits into the presynaptic neurone inhibited transmission, and also reduced the AHP amplitude. Furthermore, NA-induced inhibition was occluded by pre-injection of G subunits. The Ca2+ channel blocker Cd2+ mimicked NA effects on transmitter release. Cd2+, NA and G subunits also inhibited somatic Ca2+ current. In contrast to effects on AP-evoked transmitter release, NA had no clear action on AP-independent EPSPs induced by hypertonic solutions. These results demonstrate that G subunits functionally mediate inhibition of transmitter release by 2-adrenoceptors and represent important regulators of synaptic transmission at mammalian presynaptic terminals.
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Introduction
Activation of presynaptic GPCRs represents an important regulatory mechanism for neurotransmitter release (Miller, 1998). Presynaptic inhibition by GPCRs has been proposed variously to involve an inhibition of Ca2+ channels, an activation of K+ channels, or a direct action on the exocytotic release machinery itself (Thompson et al. 1993; Wu & Saggau, 1997; Miller, 1998). Common to all of these mechanisms is the proposed mediation by G subunits, liberated from G trimers by receptor occupation (Pierce et al. 2002). However, the functional contribution of G pathways to transmitter release from presynaptic terminals remains inferred largely from recordings of postsynaptic events. Due mainly to the lack of accessibility of most presynaptic terminals to exogenous peptides, few direct studies on G mechanism of action have been attempted. One exception amongst mammalian synapses is the calyx of Held preparation, in which unusually large, glutamatergic synapses occur in the medial nucleus of the trapezoid body (Forsythe, 1994). In these terminals, injection of G subunits was shown to inhibit Ca2+ currents and partially occlude GABAB receptor agonist effects (Kajikawa et al. 2001). However, alternative mechanisms have been proposed. Purified G subunits have been introduced directly into lamprey reticulospinal–motoneurone synapses and shown to inhibit evoked transmitter release (Blackmer et al. 2001). Here, G subunits were proposed to interact directly with the exocytotic release machinery, independently of effects on Ca2+ channels (Blackmer et al. 2001; Takahashi et al. 2001). This mechanism was proposed to involve the binding of G subunits to syntaxin and/or SNAP-25 which may prevent the fusion of synaptic vesicles (see also Spafford & Zamponi, 2003). In addition, injection of GTPS, a non-hydrolysable GTP analogue, into squid giant axon terminals was reported to inhibit synaptic transmission with few accompanying effects on intracellular Ca2+ levels or Ca2+ influx (Hess et al. 1993). Presynaptic inhibition by GPCRs independent of effects on Ca2+ influx has parallels at many central synapses, where inhibition of spontaneous, AP-independent transmitter release can be quantified as effects on ‘miniature’ postsynaptic events (Miller, 1998); such events are also mediated potentially by G subunits (Harvey & Stephens, 2004). Overall, these studies suggest potential differences in presynaptic Gmediated signalling mechanisms for the inhibition of transmitter release.
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Here, we sought to extend studies on the functional consequences of direct, presynaptic injection of G subunits by examining effects on transmitter release in a mammalian model synapse for fast cholinergic transmission formed by SCGNs in culture. We took advantage of the unique, large presynaptic cells which are amenable to injection of exogenous peptides; these peptides can diffuse rapidly to nerve terminals contacting synaptically with adjacent neurones (Mochida et al. 1996). We show that 2-adrenoceptor inhibition of AP-evoked transmitter release is mediated by G subunits and also parallels the inhibition of somatic voltage-dependent Ca2+ channels (VDCCs); in contrast, 2-adrenoceptors had no clear effects on AP-independent transmitter release. These data demonstrate directly that G subunits act as important regulators of GPCR-mediated inhibition of synaptic transmission at mammalian presynaptic terminals.
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Methods
Synaptic transmission between SCGNs
Wistar ST rats were decapitated on postnatal day 7 under diethylether anaesthesia according to the guidelines of the Physiological Society of Japan. SCGNs were isolated and maintained in culture for 5–6 weeks in a growth medium of 84% Eagle's minimal essential medium, 10% fetal calf serum, 50% horse serum, 1% penicillin–streptomycin (all from Gibco Industries Inc., Langley, OK, USA) and 25 ng ml–1 nerve growth factor (2.5 S, grade II; Alomone, Jerusalem, Israel) as described (Mochida et al. 1995). Conventional intracellular recordings were made from two neighbouring neurones using microelectrodes filled with 1 M potassium acetate (KAc; 70–90 M). Neurones were superfused with modified Krebs solution containing (mM): NaCl, 136; KCl, 5.9; CaCl2, 5.1; MgCl2, 1.2; glucose, 11; and Na-Hepes, 3; pH 7.4 with NaOH. EPSPs were recorded from one of the neurones, while APs were generated in the other neurone by passage of current (1–1.5 nA of 15 ms square pulse) through an intracellular recording electrode. The 2-adrenoceptor agents NA, clonidine or yohimbine (all from Sigma, St Louis, MO, USA), or CdCl2 (Kanto Chemical Co. Inc., Tokyo, Japan) were dissolved in modified Krebs solution and superfused or drop-applied. In experiments to block the involvement of Gi/o, SCGNs were incubated with 500 ng ml–1 PTX (Sigma) in the growth medium for 12–36 h at 37°C, 95% O2 / 5% CO2. G transducin cDNA (0.25 mg ml–1), a kind gift of Professor A. Dolphin (UCL, London, UK), was dissolved in 150 mM KAc, 5 mM Mg-ATP, 10 mM Hepes, pH 7.3 and injected into SCGN nuclei from a microglass pipette as described previously (Mochida et al. 2003). Dextran fluorescein (10 kDa, Molecular Probes, Eugene, OR, USA) was introduced via the pipette solution to monitor cDNA entry into nuclei. After incubation at 37°C, 95% O2 / 5% CO2 in the growth medium (see above) for 24–48 h, injected neurones were identified with an inverted microscope equipped with an epifluorescent unit. G subunits, a kind gift of Dr J. Pitcher (UCL), purified from bovine brain as described by Casey et al. (1989), or control buffer (20 mM Tris pH 8.0, 100 mM NaCl, 2 mM DTT, 0.1% Lubrol (Serva, Heidelberg, Germany)), were introduced into the presynaptic cell body by diffusion from a suction glass pipette for 2–3 min (GC150-F10, Harvard Apparatus, Kent, UK; 15–20 M tip resistance). Fast Green FCF (5%, Sigma) was included in the injection solution to confirm entry into the presynaptic cell. AP-independent transmitter release was induced by puff application of 0.5 M sucrose onto synaptically coupled SCGNs (Mochida et al. 1998). Electrophysiological data were collected and analysed using software written by the Late Dr Ladislav Tauc (CNRS, France). Some recordings were monitored with a strip chart recorder (Nihon Kohden, Japan).
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Patch clamp recordings in SCGNs
Whole-cell electrophysiological recordings were made from individual SCGNs, cultured for 3–4 weeks, using borosilicate glass electrodes (GC150-TF10, Harvard Apparatus) which, when filled with an intracellular solution containing (mM): caesium aspartate, 140; EGTA, 5; MgCl2, 2; CaCl2, 0.1; K2ATP, 2; and Hepes, 10; pH 7.2 with CsOH, had tip resistances between 3 and 5 M. Neurones were superfused with modified Krebs solution containing (mM): TEACl, 136; CsCl, 5.9; MgCl2, 1.2; Na-Hepes, 3; and 3 μM tetrodotoxin (Wako Chemical Co., Osaka, Japan) (pH 7.4 with NaOH), together with 1 mM BaCl2. Whole-cell currents were acquired using Clampex software via an Axopatch 200A amplifier (Axon Instruments, Union City, CA, USA). Data were filtered at 2–5 kHz, digitized at 5–20 kHz and analysed using pCLAMP and Origin (Microcal, Northampton, MA, USA) software. Series resistance was compensated between 50 and 70%. Current records are shown following leak and capacitance current subtraction (P/4 protocol) and correction for liquid junction potentials.
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All data are given as mean values ± S.E.M. and statistical significance was determined using Student's paired or unpaired t tests as appropriate.
Results
2-Adrenoceptor activation inhibits synaptic transmission in cultured SCGNs
To investigate the effects of activation of GPCRs on synaptic transmission in SCGNs cultured for 5–6 weeks, we examined the actions of NA, an agonist whose somatic actions have been characterized extensively in SCGNs in short-term culture (e.g. Schofield, 1990), and which is thought to modulate cholinergic transmission in the SCG (Libet, 1979). Transmission was examined by monitoring pre- and postsynaptic events in synaptically coupled SCGNs (Figs 1–4). Bath application of NA caused a clear reduction in evoked EPSP amplitude (Fig. 1A and D (post)). NA-induced EPSP reduction was accompanied by presynaptic effects. As illustrated in Fig. 1A (pre), the amplitude of the AHP in the AP waveform recorded in presynaptic cells was reduced reversibly by 10 μM NA (from 15.6 ± 1.4 to 10.1 ± 1.0 mV, n = 3, P < 0.05; Fig. 1Ca). The time course of NA-induced reduction in the AHP amplitude is illustrated in a representative chart recorder trace in Fig. 1D (pre), which follows membrane potential and associated AHP in response to presynaptic APs. NA typically caused a rapid reduction in AHP amplitude, which recovered slowly. NA (10 μM) also reduced reversibly the AP duration at half of peak amplitude (from 2.47 ± 0.09 to 1.73 ± 0.15 ms, n = 3, P < 0.05; Fig. 1Cb), as illustrated in a typical expanded time course of the AP waveform in Fig. 1B.
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A, action potential (AP) waveform recorded in the presynaptic superior cervical ganglion neurone (SCGN; pre) and corresponding evoked excitatory postsynaptic potential EPSP (post) under different experimental conditions. NA and Cd2+ reduced EPSP and after-hyperpolarization potential (AHP) amplitude and also abolished a recurrent EPSP (*) in this example. B, expanded AP waveform corresponding to conditions in A. Arrows indicate AP duration. C, summary bar graphs illustrating mean (± S.E.M.) decrease in AHP amplitude (a) and AP duration by (b) 100 μM Cd2+ and 10 μM NA. Con, control. *P < 0.05. D, example chart recorder traces showing pre- and postsynaptic effects of Cd2+ (upper panel) and NA (lower panel). EPSPs were evoked at a frequency of 0.1 Hz.
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The actions of NA on AP duration and AHP may be characteristic of agents that reduce Ca2+ entry into SCGNs in culture (Mochida & Kobayashi, 1986a,b). Therefore, we compared the effects of NA with 100 μM Cd2+, a general blocker of VDCCs, including CaV2.2 (N-type) channels which predominantly mediate cholinergic transmission in SCGNs (Mochida et al. 1995). Cd2+ (100 μM) greatly reduced EPSP amplitude (Fig. 1A and D (post)). Cd2+ strongly and rapidly reduced AHP amplitude from 14.7 ± 2.2 to 6.79 ± 1.4 mV (n = 5, P < 0.05) (Fig. 1A, Ca and D (pre)). Cd2+ also reduced AP duration at half of peak amplitude from 2.85 ± 0.22 to 1.93 ± 0.18 ms (n = 5, P < 0.05) (see Fig. 1B and Cb). All actions of Cd2+ were readily reversible. Together, the presynaptic actions seen were consistent with a reduction of Ca2+ influx into SCGNs. NA caused a reduction in EPSP amplitude over a range of concentrations: 200 nM (–13.8 ± 7.4%, n = 4), 1 μM (–25.1 ± 9.0%, n = 5) and 10 μM NA (–39.2 ± 6.1%, n = 10) gave a dose-related inhibition (Fig. 2A, B and E). Higher concentrations of NA (50–200 μM) caused no further inhibition (data not shown). The selective 2-adrenoceptor agonist, clonidine (10 μM), also produced a clear reduction in EPSP amplitude (–27.7 ± 6.8%, n = 6) (Fig. 2C and E). The NA-induced decrease in EPSPs was significantly reduced by the 2-adrenoceptor selective antagonist yohimbine (–12.9 ± 8.0%, P < 0.05) (Fig. 2A, D and E). Yohimbine also reduced the presynaptic effects of NA; for example, the NA-induced reduction in AHP amplitude was reversibly blocked by yohimbine (Fig. 2A (pre)). Together, these data were consistent with the activation of presynaptic 2-adrenoceptors causing an inhibition of cholinergic transmission in SCGNs.
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A, example chart recorder traces showing pre- and postsynaptic effects of NA (top panel), block of NA action by yohimbine (middle panel) and recovery of NA effects (bottom panel) recorded in the same cell. B, dose-related effects of NA on evoked EPSP amplitude; smoothed values of the normalized average EPSP with moving average algorithm plotted against time are shown. C, effects of clonidine on EPSP amplitude. Circles: averaged values of normalized EPSP. Continuous line: smoothed values of the normalized average EPSP (C and D). D, block of NA effects on EPSP amplitude by yohimbine (grouped data for yohimbine 1 μMversus 10 μM NA (3); 2 μMversus 5 μM NA (3); and 2 μMversus 10 μM NA (3). In A–D, 2-adrenoceptor agents were applied during indicated horizontal bars. Numbers in parentheses indicate number of replicants (n). EPSPs were evoked at a frequency of 0.1 Hz. E, summary bar graph illustrating mean (± S.E.M.) decrease in EPSP amplitude at 7 min after 2-adrenoceptor agent application. *P < 0.05 in comparison with effects of 10 μM NA in the same batch of cells (shown in D).
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G subunits mediate 2-adrenoceptor-induced inhibition of synaptic transmission
Agonist occupation of GPCRs releases GDP from a specific receptor-coupled G subunit which forms an activated G-GTP complex, resulting in release of free G dimers (Pierce et al. 2002). To examine the G protein contribution to NA inhibition, SCGNs were incubated with the G protein uncoupling agent PTX (500 ng ml–1, 24–48 h) (Fig. 3A and C). In PTX-treated synapses, the NA-induced reduction of EPSP amplitude was significantly attenuated to –14.5 ± 3.5% (n = 6, P < 0.01), implicating the involvement of Gi/o subunits. We next examined the effects of sequestering endogenous G subunits by expression of the Gbinding protein G transducin, in individual presynaptic cells (Fig. 3B and C). In synapses examined 24 h after nuclear injection of G transducin cDNA, 10 μM NA still produced a clear reduction in EPSP amplitude (–35.0 ± 4.5%, n = 5), not significantly different to that seen in untreated cells. In contrast, in synapses examined at least 48 h after the cDNA injection, 10 μM NA-induced reduction in EPSP amplitude was significantly attenuated to –9.0 ± 3.8% (n = 6, P < 0.01) (Fig. 3B and C). This distinction may be due to the time taken for a sufficient concentration of G transducin to be translocated into the terminals and fully sequester G subunits. Overall, these data show that G transducin produced a time-dependent attenuation of NA action and implicate G subunits as a predominant mediator of NA action. We tested this hypothesis by injecting purified G subunits (10 μM in the injection pipette) directly into the presynaptic cell of synaptically coupled SCGNs. G, injected following a minimum of 20 min stable recording of evoked EPSPs, caused a clear reduction in EPSP amplitude (–29.0 ± 7.2% at 50 min after injection, n = 6) which took more than 30 min to peak and lasted for at least 2 h of recording (Fig. 4A and D). In contrast, injection of heat-denatured G subunits had only minor effects on EPSP amplitude, with a level of inhibition (–5.5 ± 2.4% at 50 min after injection, n = 5), significantly less than that seen with G (Fig. 4D). To investigate any possible contribution of presynaptic K+ channels to inhibition of transmitter release, G effects were examined in the continued presence of 1 mM Ba2+ (a general blocker of K+ channels, including G protein-coupled inward rectifier channels, which are potential targets of Gmediated activation, Clapham & Neer, 1997). In modified Krebs solution supplemented with Ba2+, injection of G subunits (10 μM in the injection pipette) caused a clear reduction in EPSP amplitude (–30.5 ± 9.3% at 50 min after injection, n = 5), which was no different to that seen for G in normal extracellular solution (P = 0.51) (Fig. 4D).
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A, effects of 24–48 h pre-incubation with pertussis toxin (PTX) on NA-induced reduction of evoked EPSP amplitude. B, effects of 24 and 48 h expression of G transducin on NA-induced reduction of evoked EPSP amplitude. In A and B, NA was applied during indicated horizontal bars by superfusion (A) or drop-application (B). Numbers in parentheses indicate number of replicants (n). EPSPs were evoked at a frequency of 0.1 Hz. Circles: averaged values of normalized EPSP. Continuous lines: smoothed values of the normalized average EPSP. C, summary bar graph illustrating the effect of PTX and G transducin on mean (± S.E.M.) NA-induced decrease in EPSP amplitude. Effects of 10 μM NA measured at 7 min (PTX) and 25 min (G transducin). *P < 0.01 between each of the data sets indicated.
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A, effects of presynaptic injection of G subunits (10 μM in the injection pipette) on evoked EPSP amplitude (n = 6). Inset: example EPSP recording traces before and after G subunits injection. EPSPs were evoked at a frequency of 0.1 Hz. Circles: averaged values of normalized EPSP. Continuous line: smoothed values of the normalized average EPSP (A and C). B, effects of G subunits on AP waveform recorded in the presynaptic cell. Example chart recorder traces showing effects of G on AHP before (a), during (b) and after (c) protein injection (top, middle panels), and AP duration (bottom panel). C, effects of NA (drop-applied during bar) on evoked EPSP amplitude in the presence (+G, 10 μM in the injection pipette, n = 6) or absence of G subunits (–G, n = 6). D, summary bar graph illustrating effects of G subunits, G in the presence of 1 mM Ba2+ added to the modified Krebs solution, and heat-denatured G controls, on mean evoked EPSP amplitude and effects of G on mean NA-induced decrease in EPSP amplitude (± S.E.M.). Effects of 10 μM NA measured at 25 min. *P < 0.001 between each of the data sets indicated.
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G also had effects on the AP waveform. As illustrated in Fig. 4B, G caused a reduction in AHP (b*) during and immediately following injection; AHP amplitude was reduced from 14.9 ± 1.6 to 11.5 ± 1.8 mV at 3 min after starting the injection (n = 7, P < 0.01). A similar reduction was not seen following injection of the carrier buffer (8.6 ± 0.2 to 8.4 ± 0.7 mV at 3 min after starting the injection, n = 3). In contrast to actions on AHP amplitude, G injection had no clear effects on AP duration at this time point (Fig. 4B, bottom traces) (1.6 ± 0.1 to 1.7 ± 0.2 ms, n = 7).
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To confirm the contribution of G to NA action, we examined the effects of pre-injection of G subunits on NA-induced inhibition (Fig. 4C and D). Presynaptic cells were injected with G 3–5 min after starting EPSP recordings, and then, 50–60 min after injection, 10 μM NA was applied. In these synapses, the inhibitory effects of 10 μM NA on EPSP amplitude were now virtually abolished (–4.0 ± 3.8%, n = 6). This lack of effect was highly significant in comparison to time-matched controls in which 10 μM NA was added in the absence of G injection (–30.0 ± 6.1%, n = 6; P < 0.001). The occlusion of NA effects by G pre-injection is consistent with 2-adrenoceptor inhibition of synaptic transmission in SCGNs being mediated by G subunits.
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G subunits and NA inhibit somatic Ca2+ currents
Synaptic transmission in SCGNs is mediated predominantly by presynaptic CaV2.2 (N-type) channels (Mochida et al. 1995), but direct measurement of Ca2+ currents in presynaptic terminals of SCGNs is technically difficult. Therefore, we examined the effects of NA and G subunits on somatic whole-cell Ca2+ currents in SCGNs in 3–4 weeks culture. A similar approach has been used to measure somatic Ca2+ currents in SCGNs in short-term culture (typically 1–4 days), leading to extensive characterization of an 2-adrenoceptor pathway (Horn & McAfee, 1980; Galvan & Adams, 1982; Schofield, 1990; Hille, 1994; Herlitze et al. 1996; Ikeda, 1996; Garcia et al. 1998; Delmas et al. 1998, 1999). However, it has been shown that the neurotransmitter released predominantly by SCGNs changes as time in culture increases. Thus, cells release NA initially (see Koh & Hille, 1997), and then, after 1–2 weeks, they switch to releasing acetylcholine predominantly (Mochida et al. 1994). Therefore, we investigated if a membrane-delimited pathway that mediates the modulation of Ca2+ currents was active at newly formed cholinergic presynaptic terminals in the long-term SCGN cultures used here.
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The actions of the inorganic ion Cd2+, which caused a dramatic reduction in EPSP amplitude (Fig. 1A and D (post)), may suggest a role for presynaptic VDCCs in the inhibition of transmitter release. Cd2+ (100 μM) also abolished somatic Ca2+ current completely (Fig. 5A). Furthermore, 10 μM NA also caused a clear reduction in current amplitude of –32.6 ± 3.7% at +10 mV (n = 12) (see Fig. 5B). A major characteristic of agonist-induced G protein inhibition of VDCCs is a slowing in current activation kinetics, associated with G subunits binding to the channel to promote a ‘reluctant’ gating state (Bean, 1989; Patil et al. 1996). Therefore, current activation was quantified by a simple time-to-peak measurement: control values (12.4 ± 1.7 ms at +10 mV, n = 12) were significantly increased by 10 μM NA (22.4 ± 5.3 ms at +10 mV, n = 12; P < 0.05), as can be seen in Fig. 5B. Higher concentrations of NA promoted further inhibition, as also illustrated in Fig. 5B. A further characteristic of G protein inhibition of VDCCs is that it can be reversed partially by a large, depolarizing prepulse, described originally as a disruption of the voltage-dependent interaction between the G protein and the VDCC (Grassi & Lux, 1989; Bean, 1989). In cells subject to NA inhibition, application of a +120 mV prepulse caused an increase in whole-cell current amplitude and also an acceleration of activation kinetics (Fig. 5C and E). In the presence of NA, a clear increase in the ratio of peak current amplitude following the prepulse (+PP) to that before (–PP) was seen (+PP: –PP = 1.36 ± 0.08 at 0 mV, n = 5), representing a significant increase above the ratio in control cells (+PP: –PP = 1.01 ± 0.02 at 0 mV, n = 5, P < 0.05) (Fig. 5C, Db and E). The lack of facilitation seen in control cells suggests a lack of tonic modulation by endogenous G subunits. We also examined NA actions on somatic Ca2+ current in cells expressing G transducin. The NA-induced reduction of Ca2+ current amplitude was now attenuated significantly to –10.4 ± 3.2%, n = 5, P < 0.05. These data further implicate G subunits as mediators of NA effects and are in agreement with the reported attenuation of NA action on somatic Ca2+ currents by G transducin in SCGNs in short-term culture (Delmas et al. 1999). Overall, we saw no clear distinction between G transducin effects on somatic Ca2+ currents after 24 or 48 h, in comparison to actions on transmitter release. These findings suggest that extrapolation of events at the cell soma to those affecting presynaptic release may rely on a sufficient concentration of protein reaching synaptic terminals. Finally, introduction of G subunits (2–10 μM in the patch pipette) directly into SCGNs caused a clear slowing in the time course of activation of whole-cell current (Fig. 5Da). Time-to-peak was slowed dramatically to 168 ± 15 ms at +10 mV (n = 6, P < 0.001 compared to control currents). Application of a +120 mV prepulse revealed a large Ginduced reduction in current amplitude and time-to-peak (Fig. 5Da and E), with a significant increase in peak current ratio to 1.47 ± 0.15 at 0 mV, n = 5 (P < 0.05 compared to control ratio) (Fig. 5E). Overall, these data indicate that Cd2+, NA and G caused clear reductions in somatic Ca2+ currents in SCGNs in long-term culture. These actions have close parallels with the effects of the same agents on presynaptic transmitter release described above.
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Example effect of Cd2+ (A) and NA (B) on whole-cell Ca2+ currents. Currents were activated by a depolarizing step to +10 mV from a holding potential (VH) of –70 mV. C, effect of a conditioning prepulse to +120 mV (dotted line, +PP) on NA-induced inhibition of whole-cell Ca2+ currents. D, example effect of a conditioning prepulse to +120 mV (+PP) on (a) G (6.7 μM in the patch pipette)-inhibited Ca2+ currents, and (b) control currents. Currents were activated by a depolarizing step to +10 mV; VH = –70 mV. E, summary bar graph illustrating effect of NA (n = 5) and G subunits (n = 5) on control (n = 5) peak current amplitude ratio (+PP: –PP at 0 mV), mean (± S.E.M.), *P < 0.05.
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NA has no effect on AP-independent EPSPs
A further hypothesis for the mechanism of Gmediated presynaptic inhibition is that action occurs independently of effects on Ca2+ influx through VDCCs, possibly via a direct interaction with the synaptic vesicle fusion machinery (Blackmer et al. 2001). In the SCGN model synapse, Ca2+ influx is triggered by generation of APs in the presynaptic SCGN; therefore, we investigated if activation of the identified 2-adrenoceptor pathway had any effect on transmitter release in the absence of APs, which may be consistent with alternative mechanisms of GPCR inhibition. AP-independent EPSPs were stimulated by focal application of hypertonic 0.5 M sucrose solution, puff-applied for 2 s onto SCGNs, in the absence of the passage of current in presynaptic cells (Mochida et al. 1998). Bath application of 10 μM NA, following a minimum of 8 min stable baseline in the sucrose response integral, had no clear effect on AP-independent EPSPs (n = 5 separate cells, Fig. 6A and B). These data suggest that, under these conditions, NA had no clear effect on acetylcholine release in SCGNs in the absence of APs. Overall, although we cannot rule out an action on the synaptic vesicle fusion machinery contributing to NA-induced inhibition of AP-evoked transmission, we find no confirmatory evidence from studies solely on transmitter release performed in the absence of APs.
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A, effects of NA on AP-independent EPSPs induced by 0.5 M sucrose (applied from a local puff pipette during period indicated by bar); raw traces before, during and after bath NA superfusion. B, normalized mean (± S.E.M.) sucrose response integral values illustrating lack of effect of NA (n = 5).
Discussion
G subunits mediate presynaptic inhibition
Although presynaptic inhibition of synaptic transmission by GPCRs is a well-known phenomenon, there is, as yet, a lack of direct demonstrations that G subunits functionally mediate inhibition and, also, if common mechanisms exist amongst central synapses. Here, we have taken advantage of the accessibility to exogenous peptides of the large, presynaptic SCGNs that form in culture, to show that modulation of fast cholinergic transmission by presynaptic 2-adrenoceptors is mediated by G subunits. Accordingly, direct injection of purified G subunits caused an inhibition of transmission between synaptically coupled SCGNs. Inhibition took more than 30 min to peak and lasted for at least 2 h of recording (Fig. 4A), consistent with G subunits diffusing from the soma to synaptic terminals to cause inhibition. Studies have concluded that transmitter secretion from SCGNs in culture occurs almost exclusively from varicosities and terminals, rather than the cell body itself (Wakshull et al. 1979; Przywara et al. 1993; Koh & Hille, 1997). Activation of the endogenous 2-adrenoceptor pathway confirmed the role of G. Accordingly, NA-induced inhibition of evoked transmitter release was (i) sensitive to PTX, (ii) reduced in a time-dependent manner by presynaptic over-expression of G transducin, and (iii) occluded by presynaptic injection of G subunits. At concentrations shown to inhibit evoked transmission, NA also inhibited somatic VDCCs, exhibiting characteristics of Gmediated modulation (Bean, 1989; Patil et al. 1996). Thus, application of NA (i) reduced whole-cell Ca2+ current amplitude, (ii) slowed current activation kinetics, and (iii) inhibition could be reversed partially by a large, depolarizing prepulse. Finally, introduction of G subunits into presynaptic SCGNs, at concentrations similar to those that inhibited synaptic transmission, also inhibited somatic VDCCs. In the SCG, NA may be released from dendrites and modulate cholinergic presynaptic neurones (Libet, 1979). The Gmediated presynaptic inhibition via 2-adrenoceptors demonstrated here in a cholinergic model synapse should therefore reflect the NA modulation of synaptic transmission in the SCG.
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The presynaptic AP waveform
The shape of the presynaptic AP waveform is critical to the modulation of synaptic transmission, transmitter release being triggered by Ca2+ entry predominantly in the AP repolarization phase (Augustine, 1990; Wheeler et al. 1996; Sabatini & Regehr, 1997). SCGNs in culture display a mixed Na+–Ca2+ spike, followed by a long-lasting AHP (Mochida & Kobayashi, 1986a,b). Here, NA reduced both the duration and the AHP amplitude of the AP waveform. These effects are characteristic of agents that reduce Ca2+ entry into SCGNs in culture (Horn & McAfee, 1980; Mochida & Kobayashi, 1986a,b). In the present study, Cd2+ also reduced AP duration and AHP amplitude. The effects of NA and Cd2+ may therefore be attributed to inhibition of VDCCs, as also reported for somatic AP waveforms in other sensory neurones that express a mixed Na+–Ca2+ spike (Dunlap & Fischbach, 1978; Werz & MacDonald, 1983). In contrast, Cd2+ has been reported to block postsynaptic events with no concurrent effect on fast presynaptic AP waveforms (Borst et al. 1995; Sabatini & Regehr, 1997). Also, NA-mediated inhibition of glutamate release at calyx of Held synapses, which occurs via an effect on presynaptic VDCCs, proceeds without clear effects on the AP waveform (Leo & Von Gersdorff, 2002). AP duration in central presynaptic terminals is typically less than 1 ms and is not followed by a pronounced AHP (Augustine, 1990; Wheeler et al. 1996), whilst for SCGNs in culture, AP duration was 3–4 ms, followed by an AHP lasting > 150 ms under our recording conditions (Figs 1 and 4), such values being comparable for neurones in the SCG (Nishi et al. 1965). Therefore, the large Ca2+ component may contribute to differences in sensitivity of AP waveforms to VDCC blockers, and also to GPCR activation, between somatic SCGN recordings and those at central presynaptic terminals (Isaacson, 1998; Takahashi et al. 1998; Leo & Von Gersdorff, 2002). The present study suggests that synaptic transmission in sympathetic ganglia may be finely tuned by 2-adrenoceptors, since the duration of the AP modulates synaptic transmission; a broadening in AP leads to increased transmitter release and conversely a narrowing will decrease release and inhibit transmission (Wheeler et al. 1996; Geiger & Jonas, 2000).
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G subunits also had effects on the somatic AP waveform in SCGNs in culture. G caused a decrease in AHP amplitude during and immediately following protein injection (see Fig. 4B), but as it did not persist during the Gmediated inhibition of synaptic transmission, this is likely to reflect a transient action of G at the soma. In contrast, we did not see any clear effects of G on AP duration. This may suggest that actions on AP duration and AHP amplitude represent distinct processes. However, as discussed above, our results suggest that agents that act by blocking Ca2+ channels would be expected to reduce both AP duration and AHP amplitude. One possible explanation is that G may concomitantly modulate additional conductances to further affect AP duration. For example, AP duration is also sensitive to modulation of K+ channels (Geiger & Jonas, 2000). Activation of K+ channels would shorten AP duration; conversely, inhibition or inactivation of K+ conductances would broaden the AP. Conceivably, the latter action may oppose the expected AP shortening due to the inhibition in Ca2+ influx by G. In this regard, signal transduction enzymes modulated by G include phospholipase A2, phospholipase C, mitogen-activated protein kinase, and several isoforms of adenylate cyclase (Clapham & Neer, 1997). Many of these Gactivated enzymes have complex effects, including inhibition or inactivation of different types of K+ channels (e.g. Jonas & Kaczmarek, 1996), and thus have the potential to alter the shape of APs that invade nerve terminals.
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Mechanism of Gmediated presynaptic inhibition
Having demonstrated that G subunits represent an important regulator of presynaptic inhibition by 2-adrenoceptors in SCGNs in culture, we examined potential mechanisms of action. Cd2+, a divalent cation that plugs the pore of VDCCs (Cloues et al. 2000), dramatically reduced transmitter release in SCGNs, and also effectively abolished somatic Ca2+ current. Similarly, NA reduced both transmitter release and somatic Ca2+ current. We examined the effects of NA in more detail and show a reduction in whole-cell Ca2+ current amplitude and a slowing of current activation, both effects being reversed by a large, depolarizing prepulse. These actions are characteristic of Gmediated modulation (Bean, 1989; Patil et al. 1996) and G subunits, introduced via the patch electrode, also caused a prominent inhibition of Ca2+ current. It is important to stress that somatic recordings may not necessarily correlate with presynaptic events (Geiger & Jonas, 2000), and that to implicate fully a G action on VDCCs at presynaptic terminals, direct recordings from these sites should be performed. However, VDCCs present at terminals contacting synaptically with adjacent neurones have been visualized recently. These VDCCs in distinct punctate clusters (Mochida et al. 2003), would also be expected to be subject to Gmediated inhibition, given that the injected G subunits reach these terminals, as the clear reduction in synaptic transmission seen here suggests. In support, studies in which NA released from short-term SCGN cultures was detected by amperometry have concluded that inhibition of transmitter release by NA showed pharmacological and kinetic similarities to the depression of somatic Ca2+ currents (Koh & Hille, 1997); it was proposed that a membrane-delimited action of G subunits may mediate this response. Thus, whilst presynaptic recordings are technically difficult due to the small size of the terminals, here we were able to use the SCGN system to introduce G subunits into presynaptic terminals via diffusion from somatic injection sites. It is clear from our findings that G subunits (in addition to NA and Cd2+) can inhibit somatic VDCCs and functionally inhibit transmitter release.
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CaV2.2 (N-type) channels predominantly mediate fast transmission in SCGNs in long-term culture (Mochida et al. 1995). As discussed above, an 2-adrenoceptor pathway has been described extensively using somatic recordings from isolated SCGNs in short-term culture. For instance, N-type VDCCs are modulated by NA via multiple G protein subunits, using both voltage-dependent and -independent pathways in these cells (Hille, 1994). Furthermore, 2-adrenoceptors couple to Go subunits to cause a voltage-dependent, PTX-sensitive inhibition of VDCCs, and to Gi subunits to cause a largely voltage-independent, PTX-resistant inhibition, mediated potentially by different G isoforms (Delmas et al. 1999). Similarly, release of NA from short-term SCGN cultures showed partial sensitivity to PTX (Koh & Hille, 1997). We demonstrate that the 2-adrenoceptor pathway is present in long-term-cultured cholinergic SCGNs and also is of functional significance in the inhibition of synaptic transmission. NA was able to cause a membrane-delimited, voltage-dependent inhibition of VDCCs and also to inhibit transmitter release by an action mediated by G subunits. Inhibition of transmitter release was reduced significantly by PTX and also in SCGNs expressing G transducin. Furthermore, NA-induced reduction of somatic Ca2+ current was also attenuated significantly by expression of G transducin, providing additional evidence for a link between actions on transmitter release and somatic Ca2+ channels.
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These data confirm G subunits as an essential presynaptic inhibitory mediator for cholinergic synaptic transmission in sympathetic ganglia. As described above, the modulation of presynaptic transmitter release had close parallels with the modulation of somatic VDCCs; these pharmacological similarities represent correlative evidence that presynaptic inhibition may involve the same mechanism. This suggestion is supported by recordings at presynaptic terminals in the calyx of Held preparation, which demonstrated that injection of G subunits caused an inhibition of VDCCs (Kajikawa et al. 2001). However, alternative mechanisms have been proposed where Gmediated inhibition of evoked transmitter release occurs downstream of Ca2+ influx, possibly via a direct interaction with the synaptic release machinery (Blackmer et al. 2001). In relation to this general mechanism, numerous GPCRs, including 2-adrenoceptors (Starke, 2001), are reported to inhibit AP-independent ‘miniature’ postsynaptic events in mammalian synapses (Miller, 1998). Here, under conditions when the major drive for Ca2+ influx, i.e. the presynaptic AP, was removed, we saw no effect of NA on transmitter release stimulated by hypertonic sucrose application. Therefore, we have no evidence that the 2-adrenoceptor pathway identified in SCGNs had any effects on AP-independent transmitter release, although we cannot rule out fully a contribution to AP-evoked synaptic transmission.
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Overall, we provide direct functional evidence that G subunits are important regulators of GPCR inhibition of transmitter release at mammalian presynaptic terminals. Results were consistent with G dimers representing important signalling molecules for presynaptic 2-adrenoceptor-mediated inhibition of transmitter release, supporting somatic studies in SCGNs. It will be of interest in the future to determine if other ion channel pathways modulated by multiple G protein subunits in SCGNs (Hille, 1994) can activate complementary pathways.
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2 Department of Physiology, Tokyo Medical University, Tokyo 160-8402, Japan
Abstract
The activation of presynaptic G protein-coupled receptors (GPCRs) is widely reported to inhibit transmitter release; however, the lack of accessibility of many presynaptic terminals has limited direct analysis of signalling mediators. We studied GPCR-mediated inhibition of fast cholinergic transmission between superior cervical ganglion neurones (SCGNs) in culture. The adrenoceptor agonist noradrenaline (NA) caused a dose-related reduction in evoked excitatory postsynaptic potentials (EPSPs). NA-induced EPSP decrease was accompanied by effects on the presynaptic action potential (AP), reducing AP duration and amplitude of the after-hyperpolarization (AHP), without affecting the pre- and postsynaptic membrane potential. All effects of NA were blocked by yohimbine and synaptic transmission was reduced by clonidine, consistent with an action at presynaptic 2-adrenoceptors. NA-induced inhibition of transmission was sensitive to pre-incubation of SCGNs with pertussis toxin (PTX), implicating the involvement of Gi/o subunits. Expression of G transducin, an agent which sequesters G protein (G) subunits, in the presynaptic neurone caused a time-dependent attenuation of NA-induced inhibition. Injection of purified G subunits into the presynaptic neurone inhibited transmission, and also reduced the AHP amplitude. Furthermore, NA-induced inhibition was occluded by pre-injection of G subunits. The Ca2+ channel blocker Cd2+ mimicked NA effects on transmitter release. Cd2+, NA and G subunits also inhibited somatic Ca2+ current. In contrast to effects on AP-evoked transmitter release, NA had no clear action on AP-independent EPSPs induced by hypertonic solutions. These results demonstrate that G subunits functionally mediate inhibition of transmitter release by 2-adrenoceptors and represent important regulators of synaptic transmission at mammalian presynaptic terminals.
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Introduction
Activation of presynaptic GPCRs represents an important regulatory mechanism for neurotransmitter release (Miller, 1998). Presynaptic inhibition by GPCRs has been proposed variously to involve an inhibition of Ca2+ channels, an activation of K+ channels, or a direct action on the exocytotic release machinery itself (Thompson et al. 1993; Wu & Saggau, 1997; Miller, 1998). Common to all of these mechanisms is the proposed mediation by G subunits, liberated from G trimers by receptor occupation (Pierce et al. 2002). However, the functional contribution of G pathways to transmitter release from presynaptic terminals remains inferred largely from recordings of postsynaptic events. Due mainly to the lack of accessibility of most presynaptic terminals to exogenous peptides, few direct studies on G mechanism of action have been attempted. One exception amongst mammalian synapses is the calyx of Held preparation, in which unusually large, glutamatergic synapses occur in the medial nucleus of the trapezoid body (Forsythe, 1994). In these terminals, injection of G subunits was shown to inhibit Ca2+ currents and partially occlude GABAB receptor agonist effects (Kajikawa et al. 2001). However, alternative mechanisms have been proposed. Purified G subunits have been introduced directly into lamprey reticulospinal–motoneurone synapses and shown to inhibit evoked transmitter release (Blackmer et al. 2001). Here, G subunits were proposed to interact directly with the exocytotic release machinery, independently of effects on Ca2+ channels (Blackmer et al. 2001; Takahashi et al. 2001). This mechanism was proposed to involve the binding of G subunits to syntaxin and/or SNAP-25 which may prevent the fusion of synaptic vesicles (see also Spafford & Zamponi, 2003). In addition, injection of GTPS, a non-hydrolysable GTP analogue, into squid giant axon terminals was reported to inhibit synaptic transmission with few accompanying effects on intracellular Ca2+ levels or Ca2+ influx (Hess et al. 1993). Presynaptic inhibition by GPCRs independent of effects on Ca2+ influx has parallels at many central synapses, where inhibition of spontaneous, AP-independent transmitter release can be quantified as effects on ‘miniature’ postsynaptic events (Miller, 1998); such events are also mediated potentially by G subunits (Harvey & Stephens, 2004). Overall, these studies suggest potential differences in presynaptic Gmediated signalling mechanisms for the inhibition of transmitter release.
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Here, we sought to extend studies on the functional consequences of direct, presynaptic injection of G subunits by examining effects on transmitter release in a mammalian model synapse for fast cholinergic transmission formed by SCGNs in culture. We took advantage of the unique, large presynaptic cells which are amenable to injection of exogenous peptides; these peptides can diffuse rapidly to nerve terminals contacting synaptically with adjacent neurones (Mochida et al. 1996). We show that 2-adrenoceptor inhibition of AP-evoked transmitter release is mediated by G subunits and also parallels the inhibition of somatic voltage-dependent Ca2+ channels (VDCCs); in contrast, 2-adrenoceptors had no clear effects on AP-independent transmitter release. These data demonstrate directly that G subunits act as important regulators of GPCR-mediated inhibition of synaptic transmission at mammalian presynaptic terminals.
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Methods
Synaptic transmission between SCGNs
Wistar ST rats were decapitated on postnatal day 7 under diethylether anaesthesia according to the guidelines of the Physiological Society of Japan. SCGNs were isolated and maintained in culture for 5–6 weeks in a growth medium of 84% Eagle's minimal essential medium, 10% fetal calf serum, 50% horse serum, 1% penicillin–streptomycin (all from Gibco Industries Inc., Langley, OK, USA) and 25 ng ml–1 nerve growth factor (2.5 S, grade II; Alomone, Jerusalem, Israel) as described (Mochida et al. 1995). Conventional intracellular recordings were made from two neighbouring neurones using microelectrodes filled with 1 M potassium acetate (KAc; 70–90 M). Neurones were superfused with modified Krebs solution containing (mM): NaCl, 136; KCl, 5.9; CaCl2, 5.1; MgCl2, 1.2; glucose, 11; and Na-Hepes, 3; pH 7.4 with NaOH. EPSPs were recorded from one of the neurones, while APs were generated in the other neurone by passage of current (1–1.5 nA of 15 ms square pulse) through an intracellular recording electrode. The 2-adrenoceptor agents NA, clonidine or yohimbine (all from Sigma, St Louis, MO, USA), or CdCl2 (Kanto Chemical Co. Inc., Tokyo, Japan) were dissolved in modified Krebs solution and superfused or drop-applied. In experiments to block the involvement of Gi/o, SCGNs were incubated with 500 ng ml–1 PTX (Sigma) in the growth medium for 12–36 h at 37°C, 95% O2 / 5% CO2. G transducin cDNA (0.25 mg ml–1), a kind gift of Professor A. Dolphin (UCL, London, UK), was dissolved in 150 mM KAc, 5 mM Mg-ATP, 10 mM Hepes, pH 7.3 and injected into SCGN nuclei from a microglass pipette as described previously (Mochida et al. 2003). Dextran fluorescein (10 kDa, Molecular Probes, Eugene, OR, USA) was introduced via the pipette solution to monitor cDNA entry into nuclei. After incubation at 37°C, 95% O2 / 5% CO2 in the growth medium (see above) for 24–48 h, injected neurones were identified with an inverted microscope equipped with an epifluorescent unit. G subunits, a kind gift of Dr J. Pitcher (UCL), purified from bovine brain as described by Casey et al. (1989), or control buffer (20 mM Tris pH 8.0, 100 mM NaCl, 2 mM DTT, 0.1% Lubrol (Serva, Heidelberg, Germany)), were introduced into the presynaptic cell body by diffusion from a suction glass pipette for 2–3 min (GC150-F10, Harvard Apparatus, Kent, UK; 15–20 M tip resistance). Fast Green FCF (5%, Sigma) was included in the injection solution to confirm entry into the presynaptic cell. AP-independent transmitter release was induced by puff application of 0.5 M sucrose onto synaptically coupled SCGNs (Mochida et al. 1998). Electrophysiological data were collected and analysed using software written by the Late Dr Ladislav Tauc (CNRS, France). Some recordings were monitored with a strip chart recorder (Nihon Kohden, Japan).
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Patch clamp recordings in SCGNs
Whole-cell electrophysiological recordings were made from individual SCGNs, cultured for 3–4 weeks, using borosilicate glass electrodes (GC150-TF10, Harvard Apparatus) which, when filled with an intracellular solution containing (mM): caesium aspartate, 140; EGTA, 5; MgCl2, 2; CaCl2, 0.1; K2ATP, 2; and Hepes, 10; pH 7.2 with CsOH, had tip resistances between 3 and 5 M. Neurones were superfused with modified Krebs solution containing (mM): TEACl, 136; CsCl, 5.9; MgCl2, 1.2; Na-Hepes, 3; and 3 μM tetrodotoxin (Wako Chemical Co., Osaka, Japan) (pH 7.4 with NaOH), together with 1 mM BaCl2. Whole-cell currents were acquired using Clampex software via an Axopatch 200A amplifier (Axon Instruments, Union City, CA, USA). Data were filtered at 2–5 kHz, digitized at 5–20 kHz and analysed using pCLAMP and Origin (Microcal, Northampton, MA, USA) software. Series resistance was compensated between 50 and 70%. Current records are shown following leak and capacitance current subtraction (P/4 protocol) and correction for liquid junction potentials.
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All data are given as mean values ± S.E.M. and statistical significance was determined using Student's paired or unpaired t tests as appropriate.
Results
2-Adrenoceptor activation inhibits synaptic transmission in cultured SCGNs
To investigate the effects of activation of GPCRs on synaptic transmission in SCGNs cultured for 5–6 weeks, we examined the actions of NA, an agonist whose somatic actions have been characterized extensively in SCGNs in short-term culture (e.g. Schofield, 1990), and which is thought to modulate cholinergic transmission in the SCG (Libet, 1979). Transmission was examined by monitoring pre- and postsynaptic events in synaptically coupled SCGNs (Figs 1–4). Bath application of NA caused a clear reduction in evoked EPSP amplitude (Fig. 1A and D (post)). NA-induced EPSP reduction was accompanied by presynaptic effects. As illustrated in Fig. 1A (pre), the amplitude of the AHP in the AP waveform recorded in presynaptic cells was reduced reversibly by 10 μM NA (from 15.6 ± 1.4 to 10.1 ± 1.0 mV, n = 3, P < 0.05; Fig. 1Ca). The time course of NA-induced reduction in the AHP amplitude is illustrated in a representative chart recorder trace in Fig. 1D (pre), which follows membrane potential and associated AHP in response to presynaptic APs. NA typically caused a rapid reduction in AHP amplitude, which recovered slowly. NA (10 μM) also reduced reversibly the AP duration at half of peak amplitude (from 2.47 ± 0.09 to 1.73 ± 0.15 ms, n = 3, P < 0.05; Fig. 1Cb), as illustrated in a typical expanded time course of the AP waveform in Fig. 1B.
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A, action potential (AP) waveform recorded in the presynaptic superior cervical ganglion neurone (SCGN; pre) and corresponding evoked excitatory postsynaptic potential EPSP (post) under different experimental conditions. NA and Cd2+ reduced EPSP and after-hyperpolarization potential (AHP) amplitude and also abolished a recurrent EPSP (*) in this example. B, expanded AP waveform corresponding to conditions in A. Arrows indicate AP duration. C, summary bar graphs illustrating mean (± S.E.M.) decrease in AHP amplitude (a) and AP duration by (b) 100 μM Cd2+ and 10 μM NA. Con, control. *P < 0.05. D, example chart recorder traces showing pre- and postsynaptic effects of Cd2+ (upper panel) and NA (lower panel). EPSPs were evoked at a frequency of 0.1 Hz.
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The actions of NA on AP duration and AHP may be characteristic of agents that reduce Ca2+ entry into SCGNs in culture (Mochida & Kobayashi, 1986a,b). Therefore, we compared the effects of NA with 100 μM Cd2+, a general blocker of VDCCs, including CaV2.2 (N-type) channels which predominantly mediate cholinergic transmission in SCGNs (Mochida et al. 1995). Cd2+ (100 μM) greatly reduced EPSP amplitude (Fig. 1A and D (post)). Cd2+ strongly and rapidly reduced AHP amplitude from 14.7 ± 2.2 to 6.79 ± 1.4 mV (n = 5, P < 0.05) (Fig. 1A, Ca and D (pre)). Cd2+ also reduced AP duration at half of peak amplitude from 2.85 ± 0.22 to 1.93 ± 0.18 ms (n = 5, P < 0.05) (see Fig. 1B and Cb). All actions of Cd2+ were readily reversible. Together, the presynaptic actions seen were consistent with a reduction of Ca2+ influx into SCGNs. NA caused a reduction in EPSP amplitude over a range of concentrations: 200 nM (–13.8 ± 7.4%, n = 4), 1 μM (–25.1 ± 9.0%, n = 5) and 10 μM NA (–39.2 ± 6.1%, n = 10) gave a dose-related inhibition (Fig. 2A, B and E). Higher concentrations of NA (50–200 μM) caused no further inhibition (data not shown). The selective 2-adrenoceptor agonist, clonidine (10 μM), also produced a clear reduction in EPSP amplitude (–27.7 ± 6.8%, n = 6) (Fig. 2C and E). The NA-induced decrease in EPSPs was significantly reduced by the 2-adrenoceptor selective antagonist yohimbine (–12.9 ± 8.0%, P < 0.05) (Fig. 2A, D and E). Yohimbine also reduced the presynaptic effects of NA; for example, the NA-induced reduction in AHP amplitude was reversibly blocked by yohimbine (Fig. 2A (pre)). Together, these data were consistent with the activation of presynaptic 2-adrenoceptors causing an inhibition of cholinergic transmission in SCGNs.
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A, example chart recorder traces showing pre- and postsynaptic effects of NA (top panel), block of NA action by yohimbine (middle panel) and recovery of NA effects (bottom panel) recorded in the same cell. B, dose-related effects of NA on evoked EPSP amplitude; smoothed values of the normalized average EPSP with moving average algorithm plotted against time are shown. C, effects of clonidine on EPSP amplitude. Circles: averaged values of normalized EPSP. Continuous line: smoothed values of the normalized average EPSP (C and D). D, block of NA effects on EPSP amplitude by yohimbine (grouped data for yohimbine 1 μMversus 10 μM NA (3); 2 μMversus 5 μM NA (3); and 2 μMversus 10 μM NA (3). In A–D, 2-adrenoceptor agents were applied during indicated horizontal bars. Numbers in parentheses indicate number of replicants (n). EPSPs were evoked at a frequency of 0.1 Hz. E, summary bar graph illustrating mean (± S.E.M.) decrease in EPSP amplitude at 7 min after 2-adrenoceptor agent application. *P < 0.05 in comparison with effects of 10 μM NA in the same batch of cells (shown in D).
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G subunits mediate 2-adrenoceptor-induced inhibition of synaptic transmission
Agonist occupation of GPCRs releases GDP from a specific receptor-coupled G subunit which forms an activated G-GTP complex, resulting in release of free G dimers (Pierce et al. 2002). To examine the G protein contribution to NA inhibition, SCGNs were incubated with the G protein uncoupling agent PTX (500 ng ml–1, 24–48 h) (Fig. 3A and C). In PTX-treated synapses, the NA-induced reduction of EPSP amplitude was significantly attenuated to –14.5 ± 3.5% (n = 6, P < 0.01), implicating the involvement of Gi/o subunits. We next examined the effects of sequestering endogenous G subunits by expression of the Gbinding protein G transducin, in individual presynaptic cells (Fig. 3B and C). In synapses examined 24 h after nuclear injection of G transducin cDNA, 10 μM NA still produced a clear reduction in EPSP amplitude (–35.0 ± 4.5%, n = 5), not significantly different to that seen in untreated cells. In contrast, in synapses examined at least 48 h after the cDNA injection, 10 μM NA-induced reduction in EPSP amplitude was significantly attenuated to –9.0 ± 3.8% (n = 6, P < 0.01) (Fig. 3B and C). This distinction may be due to the time taken for a sufficient concentration of G transducin to be translocated into the terminals and fully sequester G subunits. Overall, these data show that G transducin produced a time-dependent attenuation of NA action and implicate G subunits as a predominant mediator of NA action. We tested this hypothesis by injecting purified G subunits (10 μM in the injection pipette) directly into the presynaptic cell of synaptically coupled SCGNs. G, injected following a minimum of 20 min stable recording of evoked EPSPs, caused a clear reduction in EPSP amplitude (–29.0 ± 7.2% at 50 min after injection, n = 6) which took more than 30 min to peak and lasted for at least 2 h of recording (Fig. 4A and D). In contrast, injection of heat-denatured G subunits had only minor effects on EPSP amplitude, with a level of inhibition (–5.5 ± 2.4% at 50 min after injection, n = 5), significantly less than that seen with G (Fig. 4D). To investigate any possible contribution of presynaptic K+ channels to inhibition of transmitter release, G effects were examined in the continued presence of 1 mM Ba2+ (a general blocker of K+ channels, including G protein-coupled inward rectifier channels, which are potential targets of Gmediated activation, Clapham & Neer, 1997). In modified Krebs solution supplemented with Ba2+, injection of G subunits (10 μM in the injection pipette) caused a clear reduction in EPSP amplitude (–30.5 ± 9.3% at 50 min after injection, n = 5), which was no different to that seen for G in normal extracellular solution (P = 0.51) (Fig. 4D).
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A, effects of 24–48 h pre-incubation with pertussis toxin (PTX) on NA-induced reduction of evoked EPSP amplitude. B, effects of 24 and 48 h expression of G transducin on NA-induced reduction of evoked EPSP amplitude. In A and B, NA was applied during indicated horizontal bars by superfusion (A) or drop-application (B). Numbers in parentheses indicate number of replicants (n). EPSPs were evoked at a frequency of 0.1 Hz. Circles: averaged values of normalized EPSP. Continuous lines: smoothed values of the normalized average EPSP. C, summary bar graph illustrating the effect of PTX and G transducin on mean (± S.E.M.) NA-induced decrease in EPSP amplitude. Effects of 10 μM NA measured at 7 min (PTX) and 25 min (G transducin). *P < 0.01 between each of the data sets indicated.
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A, effects of presynaptic injection of G subunits (10 μM in the injection pipette) on evoked EPSP amplitude (n = 6). Inset: example EPSP recording traces before and after G subunits injection. EPSPs were evoked at a frequency of 0.1 Hz. Circles: averaged values of normalized EPSP. Continuous line: smoothed values of the normalized average EPSP (A and C). B, effects of G subunits on AP waveform recorded in the presynaptic cell. Example chart recorder traces showing effects of G on AHP before (a), during (b) and after (c) protein injection (top, middle panels), and AP duration (bottom panel). C, effects of NA (drop-applied during bar) on evoked EPSP amplitude in the presence (+G, 10 μM in the injection pipette, n = 6) or absence of G subunits (–G, n = 6). D, summary bar graph illustrating effects of G subunits, G in the presence of 1 mM Ba2+ added to the modified Krebs solution, and heat-denatured G controls, on mean evoked EPSP amplitude and effects of G on mean NA-induced decrease in EPSP amplitude (± S.E.M.). Effects of 10 μM NA measured at 25 min. *P < 0.001 between each of the data sets indicated.
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G also had effects on the AP waveform. As illustrated in Fig. 4B, G caused a reduction in AHP (b*) during and immediately following injection; AHP amplitude was reduced from 14.9 ± 1.6 to 11.5 ± 1.8 mV at 3 min after starting the injection (n = 7, P < 0.01). A similar reduction was not seen following injection of the carrier buffer (8.6 ± 0.2 to 8.4 ± 0.7 mV at 3 min after starting the injection, n = 3). In contrast to actions on AHP amplitude, G injection had no clear effects on AP duration at this time point (Fig. 4B, bottom traces) (1.6 ± 0.1 to 1.7 ± 0.2 ms, n = 7).
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To confirm the contribution of G to NA action, we examined the effects of pre-injection of G subunits on NA-induced inhibition (Fig. 4C and D). Presynaptic cells were injected with G 3–5 min after starting EPSP recordings, and then, 50–60 min after injection, 10 μM NA was applied. In these synapses, the inhibitory effects of 10 μM NA on EPSP amplitude were now virtually abolished (–4.0 ± 3.8%, n = 6). This lack of effect was highly significant in comparison to time-matched controls in which 10 μM NA was added in the absence of G injection (–30.0 ± 6.1%, n = 6; P < 0.001). The occlusion of NA effects by G pre-injection is consistent with 2-adrenoceptor inhibition of synaptic transmission in SCGNs being mediated by G subunits.
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G subunits and NA inhibit somatic Ca2+ currents
Synaptic transmission in SCGNs is mediated predominantly by presynaptic CaV2.2 (N-type) channels (Mochida et al. 1995), but direct measurement of Ca2+ currents in presynaptic terminals of SCGNs is technically difficult. Therefore, we examined the effects of NA and G subunits on somatic whole-cell Ca2+ currents in SCGNs in 3–4 weeks culture. A similar approach has been used to measure somatic Ca2+ currents in SCGNs in short-term culture (typically 1–4 days), leading to extensive characterization of an 2-adrenoceptor pathway (Horn & McAfee, 1980; Galvan & Adams, 1982; Schofield, 1990; Hille, 1994; Herlitze et al. 1996; Ikeda, 1996; Garcia et al. 1998; Delmas et al. 1998, 1999). However, it has been shown that the neurotransmitter released predominantly by SCGNs changes as time in culture increases. Thus, cells release NA initially (see Koh & Hille, 1997), and then, after 1–2 weeks, they switch to releasing acetylcholine predominantly (Mochida et al. 1994). Therefore, we investigated if a membrane-delimited pathway that mediates the modulation of Ca2+ currents was active at newly formed cholinergic presynaptic terminals in the long-term SCGN cultures used here.
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The actions of the inorganic ion Cd2+, which caused a dramatic reduction in EPSP amplitude (Fig. 1A and D (post)), may suggest a role for presynaptic VDCCs in the inhibition of transmitter release. Cd2+ (100 μM) also abolished somatic Ca2+ current completely (Fig. 5A). Furthermore, 10 μM NA also caused a clear reduction in current amplitude of –32.6 ± 3.7% at +10 mV (n = 12) (see Fig. 5B). A major characteristic of agonist-induced G protein inhibition of VDCCs is a slowing in current activation kinetics, associated with G subunits binding to the channel to promote a ‘reluctant’ gating state (Bean, 1989; Patil et al. 1996). Therefore, current activation was quantified by a simple time-to-peak measurement: control values (12.4 ± 1.7 ms at +10 mV, n = 12) were significantly increased by 10 μM NA (22.4 ± 5.3 ms at +10 mV, n = 12; P < 0.05), as can be seen in Fig. 5B. Higher concentrations of NA promoted further inhibition, as also illustrated in Fig. 5B. A further characteristic of G protein inhibition of VDCCs is that it can be reversed partially by a large, depolarizing prepulse, described originally as a disruption of the voltage-dependent interaction between the G protein and the VDCC (Grassi & Lux, 1989; Bean, 1989). In cells subject to NA inhibition, application of a +120 mV prepulse caused an increase in whole-cell current amplitude and also an acceleration of activation kinetics (Fig. 5C and E). In the presence of NA, a clear increase in the ratio of peak current amplitude following the prepulse (+PP) to that before (–PP) was seen (+PP: –PP = 1.36 ± 0.08 at 0 mV, n = 5), representing a significant increase above the ratio in control cells (+PP: –PP = 1.01 ± 0.02 at 0 mV, n = 5, P < 0.05) (Fig. 5C, Db and E). The lack of facilitation seen in control cells suggests a lack of tonic modulation by endogenous G subunits. We also examined NA actions on somatic Ca2+ current in cells expressing G transducin. The NA-induced reduction of Ca2+ current amplitude was now attenuated significantly to –10.4 ± 3.2%, n = 5, P < 0.05. These data further implicate G subunits as mediators of NA effects and are in agreement with the reported attenuation of NA action on somatic Ca2+ currents by G transducin in SCGNs in short-term culture (Delmas et al. 1999). Overall, we saw no clear distinction between G transducin effects on somatic Ca2+ currents after 24 or 48 h, in comparison to actions on transmitter release. These findings suggest that extrapolation of events at the cell soma to those affecting presynaptic release may rely on a sufficient concentration of protein reaching synaptic terminals. Finally, introduction of G subunits (2–10 μM in the patch pipette) directly into SCGNs caused a clear slowing in the time course of activation of whole-cell current (Fig. 5Da). Time-to-peak was slowed dramatically to 168 ± 15 ms at +10 mV (n = 6, P < 0.001 compared to control currents). Application of a +120 mV prepulse revealed a large Ginduced reduction in current amplitude and time-to-peak (Fig. 5Da and E), with a significant increase in peak current ratio to 1.47 ± 0.15 at 0 mV, n = 5 (P < 0.05 compared to control ratio) (Fig. 5E). Overall, these data indicate that Cd2+, NA and G caused clear reductions in somatic Ca2+ currents in SCGNs in long-term culture. These actions have close parallels with the effects of the same agents on presynaptic transmitter release described above.
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Example effect of Cd2+ (A) and NA (B) on whole-cell Ca2+ currents. Currents were activated by a depolarizing step to +10 mV from a holding potential (VH) of –70 mV. C, effect of a conditioning prepulse to +120 mV (dotted line, +PP) on NA-induced inhibition of whole-cell Ca2+ currents. D, example effect of a conditioning prepulse to +120 mV (+PP) on (a) G (6.7 μM in the patch pipette)-inhibited Ca2+ currents, and (b) control currents. Currents were activated by a depolarizing step to +10 mV; VH = –70 mV. E, summary bar graph illustrating effect of NA (n = 5) and G subunits (n = 5) on control (n = 5) peak current amplitude ratio (+PP: –PP at 0 mV), mean (± S.E.M.), *P < 0.05.
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NA has no effect on AP-independent EPSPs
A further hypothesis for the mechanism of Gmediated presynaptic inhibition is that action occurs independently of effects on Ca2+ influx through VDCCs, possibly via a direct interaction with the synaptic vesicle fusion machinery (Blackmer et al. 2001). In the SCGN model synapse, Ca2+ influx is triggered by generation of APs in the presynaptic SCGN; therefore, we investigated if activation of the identified 2-adrenoceptor pathway had any effect on transmitter release in the absence of APs, which may be consistent with alternative mechanisms of GPCR inhibition. AP-independent EPSPs were stimulated by focal application of hypertonic 0.5 M sucrose solution, puff-applied for 2 s onto SCGNs, in the absence of the passage of current in presynaptic cells (Mochida et al. 1998). Bath application of 10 μM NA, following a minimum of 8 min stable baseline in the sucrose response integral, had no clear effect on AP-independent EPSPs (n = 5 separate cells, Fig. 6A and B). These data suggest that, under these conditions, NA had no clear effect on acetylcholine release in SCGNs in the absence of APs. Overall, although we cannot rule out an action on the synaptic vesicle fusion machinery contributing to NA-induced inhibition of AP-evoked transmission, we find no confirmatory evidence from studies solely on transmitter release performed in the absence of APs.
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A, effects of NA on AP-independent EPSPs induced by 0.5 M sucrose (applied from a local puff pipette during period indicated by bar); raw traces before, during and after bath NA superfusion. B, normalized mean (± S.E.M.) sucrose response integral values illustrating lack of effect of NA (n = 5).
Discussion
G subunits mediate presynaptic inhibition
Although presynaptic inhibition of synaptic transmission by GPCRs is a well-known phenomenon, there is, as yet, a lack of direct demonstrations that G subunits functionally mediate inhibition and, also, if common mechanisms exist amongst central synapses. Here, we have taken advantage of the accessibility to exogenous peptides of the large, presynaptic SCGNs that form in culture, to show that modulation of fast cholinergic transmission by presynaptic 2-adrenoceptors is mediated by G subunits. Accordingly, direct injection of purified G subunits caused an inhibition of transmission between synaptically coupled SCGNs. Inhibition took more than 30 min to peak and lasted for at least 2 h of recording (Fig. 4A), consistent with G subunits diffusing from the soma to synaptic terminals to cause inhibition. Studies have concluded that transmitter secretion from SCGNs in culture occurs almost exclusively from varicosities and terminals, rather than the cell body itself (Wakshull et al. 1979; Przywara et al. 1993; Koh & Hille, 1997). Activation of the endogenous 2-adrenoceptor pathway confirmed the role of G. Accordingly, NA-induced inhibition of evoked transmitter release was (i) sensitive to PTX, (ii) reduced in a time-dependent manner by presynaptic over-expression of G transducin, and (iii) occluded by presynaptic injection of G subunits. At concentrations shown to inhibit evoked transmission, NA also inhibited somatic VDCCs, exhibiting characteristics of Gmediated modulation (Bean, 1989; Patil et al. 1996). Thus, application of NA (i) reduced whole-cell Ca2+ current amplitude, (ii) slowed current activation kinetics, and (iii) inhibition could be reversed partially by a large, depolarizing prepulse. Finally, introduction of G subunits into presynaptic SCGNs, at concentrations similar to those that inhibited synaptic transmission, also inhibited somatic VDCCs. In the SCG, NA may be released from dendrites and modulate cholinergic presynaptic neurones (Libet, 1979). The Gmediated presynaptic inhibition via 2-adrenoceptors demonstrated here in a cholinergic model synapse should therefore reflect the NA modulation of synaptic transmission in the SCG.
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The presynaptic AP waveform
The shape of the presynaptic AP waveform is critical to the modulation of synaptic transmission, transmitter release being triggered by Ca2+ entry predominantly in the AP repolarization phase (Augustine, 1990; Wheeler et al. 1996; Sabatini & Regehr, 1997). SCGNs in culture display a mixed Na+–Ca2+ spike, followed by a long-lasting AHP (Mochida & Kobayashi, 1986a,b). Here, NA reduced both the duration and the AHP amplitude of the AP waveform. These effects are characteristic of agents that reduce Ca2+ entry into SCGNs in culture (Horn & McAfee, 1980; Mochida & Kobayashi, 1986a,b). In the present study, Cd2+ also reduced AP duration and AHP amplitude. The effects of NA and Cd2+ may therefore be attributed to inhibition of VDCCs, as also reported for somatic AP waveforms in other sensory neurones that express a mixed Na+–Ca2+ spike (Dunlap & Fischbach, 1978; Werz & MacDonald, 1983). In contrast, Cd2+ has been reported to block postsynaptic events with no concurrent effect on fast presynaptic AP waveforms (Borst et al. 1995; Sabatini & Regehr, 1997). Also, NA-mediated inhibition of glutamate release at calyx of Held synapses, which occurs via an effect on presynaptic VDCCs, proceeds without clear effects on the AP waveform (Leo & Von Gersdorff, 2002). AP duration in central presynaptic terminals is typically less than 1 ms and is not followed by a pronounced AHP (Augustine, 1990; Wheeler et al. 1996), whilst for SCGNs in culture, AP duration was 3–4 ms, followed by an AHP lasting > 150 ms under our recording conditions (Figs 1 and 4), such values being comparable for neurones in the SCG (Nishi et al. 1965). Therefore, the large Ca2+ component may contribute to differences in sensitivity of AP waveforms to VDCC blockers, and also to GPCR activation, between somatic SCGN recordings and those at central presynaptic terminals (Isaacson, 1998; Takahashi et al. 1998; Leo & Von Gersdorff, 2002). The present study suggests that synaptic transmission in sympathetic ganglia may be finely tuned by 2-adrenoceptors, since the duration of the AP modulates synaptic transmission; a broadening in AP leads to increased transmitter release and conversely a narrowing will decrease release and inhibit transmission (Wheeler et al. 1996; Geiger & Jonas, 2000).
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G subunits also had effects on the somatic AP waveform in SCGNs in culture. G caused a decrease in AHP amplitude during and immediately following protein injection (see Fig. 4B), but as it did not persist during the Gmediated inhibition of synaptic transmission, this is likely to reflect a transient action of G at the soma. In contrast, we did not see any clear effects of G on AP duration. This may suggest that actions on AP duration and AHP amplitude represent distinct processes. However, as discussed above, our results suggest that agents that act by blocking Ca2+ channels would be expected to reduce both AP duration and AHP amplitude. One possible explanation is that G may concomitantly modulate additional conductances to further affect AP duration. For example, AP duration is also sensitive to modulation of K+ channels (Geiger & Jonas, 2000). Activation of K+ channels would shorten AP duration; conversely, inhibition or inactivation of K+ conductances would broaden the AP. Conceivably, the latter action may oppose the expected AP shortening due to the inhibition in Ca2+ influx by G. In this regard, signal transduction enzymes modulated by G include phospholipase A2, phospholipase C, mitogen-activated protein kinase, and several isoforms of adenylate cyclase (Clapham & Neer, 1997). Many of these Gactivated enzymes have complex effects, including inhibition or inactivation of different types of K+ channels (e.g. Jonas & Kaczmarek, 1996), and thus have the potential to alter the shape of APs that invade nerve terminals.
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Mechanism of Gmediated presynaptic inhibition
Having demonstrated that G subunits represent an important regulator of presynaptic inhibition by 2-adrenoceptors in SCGNs in culture, we examined potential mechanisms of action. Cd2+, a divalent cation that plugs the pore of VDCCs (Cloues et al. 2000), dramatically reduced transmitter release in SCGNs, and also effectively abolished somatic Ca2+ current. Similarly, NA reduced both transmitter release and somatic Ca2+ current. We examined the effects of NA in more detail and show a reduction in whole-cell Ca2+ current amplitude and a slowing of current activation, both effects being reversed by a large, depolarizing prepulse. These actions are characteristic of Gmediated modulation (Bean, 1989; Patil et al. 1996) and G subunits, introduced via the patch electrode, also caused a prominent inhibition of Ca2+ current. It is important to stress that somatic recordings may not necessarily correlate with presynaptic events (Geiger & Jonas, 2000), and that to implicate fully a G action on VDCCs at presynaptic terminals, direct recordings from these sites should be performed. However, VDCCs present at terminals contacting synaptically with adjacent neurones have been visualized recently. These VDCCs in distinct punctate clusters (Mochida et al. 2003), would also be expected to be subject to Gmediated inhibition, given that the injected G subunits reach these terminals, as the clear reduction in synaptic transmission seen here suggests. In support, studies in which NA released from short-term SCGN cultures was detected by amperometry have concluded that inhibition of transmitter release by NA showed pharmacological and kinetic similarities to the depression of somatic Ca2+ currents (Koh & Hille, 1997); it was proposed that a membrane-delimited action of G subunits may mediate this response. Thus, whilst presynaptic recordings are technically difficult due to the small size of the terminals, here we were able to use the SCGN system to introduce G subunits into presynaptic terminals via diffusion from somatic injection sites. It is clear from our findings that G subunits (in addition to NA and Cd2+) can inhibit somatic VDCCs and functionally inhibit transmitter release.
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CaV2.2 (N-type) channels predominantly mediate fast transmission in SCGNs in long-term culture (Mochida et al. 1995). As discussed above, an 2-adrenoceptor pathway has been described extensively using somatic recordings from isolated SCGNs in short-term culture. For instance, N-type VDCCs are modulated by NA via multiple G protein subunits, using both voltage-dependent and -independent pathways in these cells (Hille, 1994). Furthermore, 2-adrenoceptors couple to Go subunits to cause a voltage-dependent, PTX-sensitive inhibition of VDCCs, and to Gi subunits to cause a largely voltage-independent, PTX-resistant inhibition, mediated potentially by different G isoforms (Delmas et al. 1999). Similarly, release of NA from short-term SCGN cultures showed partial sensitivity to PTX (Koh & Hille, 1997). We demonstrate that the 2-adrenoceptor pathway is present in long-term-cultured cholinergic SCGNs and also is of functional significance in the inhibition of synaptic transmission. NA was able to cause a membrane-delimited, voltage-dependent inhibition of VDCCs and also to inhibit transmitter release by an action mediated by G subunits. Inhibition of transmitter release was reduced significantly by PTX and also in SCGNs expressing G transducin. Furthermore, NA-induced reduction of somatic Ca2+ current was also attenuated significantly by expression of G transducin, providing additional evidence for a link between actions on transmitter release and somatic Ca2+ channels.
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These data confirm G subunits as an essential presynaptic inhibitory mediator for cholinergic synaptic transmission in sympathetic ganglia. As described above, the modulation of presynaptic transmitter release had close parallels with the modulation of somatic VDCCs; these pharmacological similarities represent correlative evidence that presynaptic inhibition may involve the same mechanism. This suggestion is supported by recordings at presynaptic terminals in the calyx of Held preparation, which demonstrated that injection of G subunits caused an inhibition of VDCCs (Kajikawa et al. 2001). However, alternative mechanisms have been proposed where Gmediated inhibition of evoked transmitter release occurs downstream of Ca2+ influx, possibly via a direct interaction with the synaptic release machinery (Blackmer et al. 2001). In relation to this general mechanism, numerous GPCRs, including 2-adrenoceptors (Starke, 2001), are reported to inhibit AP-independent ‘miniature’ postsynaptic events in mammalian synapses (Miller, 1998). Here, under conditions when the major drive for Ca2+ influx, i.e. the presynaptic AP, was removed, we saw no effect of NA on transmitter release stimulated by hypertonic sucrose application. Therefore, we have no evidence that the 2-adrenoceptor pathway identified in SCGNs had any effects on AP-independent transmitter release, although we cannot rule out fully a contribution to AP-evoked synaptic transmission.
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Overall, we provide direct functional evidence that G subunits are important regulators of GPCR inhibition of transmitter release at mammalian presynaptic terminals. Results were consistent with G dimers representing important signalling molecules for presynaptic 2-adrenoceptor-mediated inhibition of transmitter release, supporting somatic studies in SCGNs. It will be of interest in the future to determine if other ion channel pathways modulated by multiple G protein subunits in SCGNs (Hille, 1994) can activate complementary pathways.
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