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J Physiol (2003), 553.2, pp. 415-426
© Copyright 2003 The Physiological Society
DOI: 10.1113/jphysiol.2003.048371

Adenosine A₁ receptor-mediated presynaptic inhibition at the calyx of Held of immature rats

Masahiro Kimura, Naoto Saitoh and Tomoyuki Takahashi

	ABSTRACT

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At the calyx of Held synapse in brainstem slices of 5- to 7-day-old (P5-7) rats, adenosine, or the type 1 adenosine (A₁) receptor agonist N⁶-cyclopentyladenosine (CPA), inhibited excitatory postsynaptic currents (EPSCs) without affecting the amplitude of miniature EPSCs. The A₁ receptor antagonist 8-cyclopentyltheophylline (CPT) had no effect on the amplitude of EPSCs evoked at a low frequency, but significantly reduced the magnitude of synaptic depression caused by repetitive stimulation at 10 Hz, suggesting that endogenous adenosine is involved in the regulation of transmitter release. Adenosine inhibited presynaptic Ca²⁺ currents (I_pCa) recorded directly from calyceal terminals, but had no effect on presynaptic K⁺ currents. When EPSCs were evoked by I_pCa during simultaneous pre- and postsynaptic recordings, the magnitude of the adenosine-induced inhibition of I_pCa fully explained that of EPSCs, suggesting that the presynaptic Ca²⁺ channel is the main target of A₁ receptors. Whereas the N-type Ca²⁺ channel blocker -conotoxin attenuated EPSCs, it had no effect on the magnitude of adenosine-induced inhibition of EPSCs. During postnatal development, in parallel with a decrease in the A₁ receptor immunoreactivity at the calyceal terminal, the inhibitory effect of adenosine became weaker. We conclude that presynaptic A₁ receptors at the immature calyx of Held synapse play a regulatory role in transmitter release during high frequency transmission, by inhibiting multiple types of presynaptic Ca²⁺ channels.

(Received 2 June 2003; accepted after revision 2 September 2003; first published online 5 September 2003)
Corresponding author T. Takahashi: Department of Neurophysiology, University of Tokyo Graduate School of Medicine, Hongo 7-3-1, Tokyo 113-0033, Japan. Email: ttakahas-tky{at}umin.ac.jp

	INTRODUCTION

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Adenosine receptors are GTP binding (G) protein-coupled receptors (GPCRs) widely expressed at nerve terminals and postsynaptic cells. Endogenous adenosine is rapidly converted from ATP (Dunwiddie et al. 1997), which is released from nerve terminals (Silinsky & Hubbard, 1973; Jo & Schlichter, 1999), neuronal somata, or glia (Latini & Pedata, 2001) in response to neuronal activity. Externally applied adenosine inhibits synaptic responses (Ginsborg & Hirst, 1972; Phillis et al. 1979; Okada & Kuroda, 1980; Dunwiddie & Haas, 1985) via activation of presynaptic A₁ receptors (Thompson et al. 1992; Yawo & Chuhma, 1993; Umemiya & Berger, 1994; Barnes-Davies & Forsythe, 1995; Bagley et al. 1999; Arrigoni et al. 2001). Ambient adenosine can regulate transmitter release, at least at some synapses (Arrigoni et al. 2001), and an increase of endogenous adenosine in the extracellular space during synaptic activation contributes to synaptic depression (Redman & Silinsky, 1994; Oliet & Poulain, 1999). Adenosine can reduce Ca²⁺ influx into the nerve terminal, by selectively inhibiting N-type Ca²⁺ channels (Yawo & Chuhma, 1993; Wu & Saggau, 1994; Umemiya & Berger, 1994). Adenosine can also activate K⁺ conductance, as reported in neuronal somata (Trussell & Jackson, 1985; Greene & Haas, 1985; Gerber et al. 1989). Through inhibition of protein kinase A (Bagley et al. 1999), adenosine may also inhibit exocytotic machinery downstream of Ca²⁺ influx (Scholtz & Miller, 1992; Silinsky & Solsona, 1992). Thus the exact mechanism by which adenosine induces presynaptic inhibition remains controversial.

The calyx of Held is a giant nerve terminal in the mammalian auditory brainstem forming an axo-somatic glutamatergic synapse onto a postsynaptic principal cell in the medial nucleus of the trapezoid body (MNTB) (Held, 1893). At this synapse, the metabotropic glutamate receptor (mGluR) agonist L-AP4, the GABA_B receptor agonist baclofen, or adenosine can presynaptically attenuate synaptic transmission (Barnes-Davies & Forsythe, 1995). Baclofen and L-AP4 inhibit presynaptic Ca²⁺ currents without affecting K⁺ currents, and their inhibitory effect on EPSCS can be fully explained by the reduction in Ca²⁺ influx (Takahashi et al. 1996, 1998). Thus adenosine receptors may share a common mechanism for the presynaptic inhibition with GABA_B receptors and mGluRs.

At the calyx of Held synapse, presynaptic Ca²⁺ channel subtypes switch from N-, P/Q- and R-types to predominantly P/Q-types during the second postnatal week (Iwasaki & Takahashi, 1998; Iwasaki et al. 2000). This change might affect adenosine-induced presynaptic inhibition if adenosine receptors are selectively coupled with N-type Ca²⁺ channels (Yawo & Chuhma, 1993; Wu & Saggau, 1994; Umemiya & Berger, 1994). Although adenosine inhibits EPSCs at the calyx of Held in P6-12 rats (Barnes-Davies & Forsythe, 1995), it is not known whether this effect remains functional throughout postnatal development. In this study we determined the target of presynaptic adenosine A₁ receptors at the calyx of Held, and examined the developmental profile of adenosine-induced presynaptic inhibition.

	METHODS

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Preparation and solutions

All experimental procedures were performed in accordance with the animal welfare guidelines of the Physiological Society of Japan. Wistar rats from P5 to P21 were decapitated under halothane anaesthesia (Forsythe & Barnes-Davies, 1993). Brainstem slices (200-400 µm thick) were cut using a tissue slicer (Leica, VT1000S). The solution for cutting tissue contained (mM); 250 sucrose, 2.5 KCl, 26 NaHCO₃, 10 glucose, 1.25 NaH₂PO₄, 1 CaCl₂, 4 MgCl₂, 0.3 myo-inositol, 2 sodium pyruvate and 0.5 ascorbic acid (pH 7.4 when bubbled with 5 % CO₂ and 95 % O₂). Slices were incubated for 30 min at 35-37 °C and maintained thereafter at room temperature in artificial cerebrospinal fluid (aCSF) bubbled with 95 % O₂ and 5 % CO₂. The standard aCSF for superfusion contained (mM): 120 NaCl, 2.5 KCl, 26 NaHCO₃, 1.25 NaH₂PO₄, 2 CaCl₂, 1 MgCl_2, 10 D-glucose, 3 myo-inositol, 2 sodium pyruvate, and 0.5 ascorbate; pH was adjusted to 7.4 when saturated with 5 % CO₂ and 95 % O₂. The superfusate routinely contained bicuculline methiodide (10 µM, Sigma) and strychnine hydrochloride (0.5 µM, Sigma) to block inhibitory synaptic responses. The postsynaptic pipette solution contained (mM): 110 CsF, 30 CsCl, 10 Hepes, 5 EGTA and 1 MgCl₂ (pH 7.4, adjusted with CsOH), and also N-(2,6-diethylphenylcarbamoylmethyl)-triethyl-ammonium chloride (QX-314; 5 mM) to block action potential generation. For recording presynaptic Ca²⁺ currents, TTX (1 µM) and tetraethylammonium (TEA) chloride (10 mM) were added to the aCSF, and the pipette solution contained (mM): CsCl 110, Hepes 40, TEA-Cl 10, EGTA 0.5, MgCl₂ 1, sodium phosphocreatinine 12, ATP-Mg 2 and GTP 0.5.-For recording presynaptic K⁺ currents, TTX (1 µM) was added to the aCSF, and the pipette solution contained (mM): potassium gluconate 97.5, KCl 32.5, Hepes 10, EGTA 5, MgCl₂ 1, sodium phosphocreatinine 12, ATP-Mg 2 and GTP 0.5.

Recording, data acquisition and analysis

The principal neurons in the medial nucleus of the trapezoid body (MNTB) were visually identified using 40 (Zeiss) or 60 (Olympus) water immersion objectives attached to an upright microscope (Axioskop, Zeiss). The whole-cell pipette resistance was 1.5-4 M for postsynaptic recordings and 4-5 M for presynaptic recordings. Series resistance was 5-12 M and 8-16 M for post- and presynaptic recordings, respectively. The recording chamber was continuously superfused with the aCSF at a flow rate of 2.0-2.5 ml min^-1. Whole-cell voltage-clamp recordings were made from MNTB neurons at a holding potential of -70 mV, and from presynaptic calyceal terminals at a holding potential of -80 mV. EPSCs were evoked by extracellular stimulation of presynaptic axons with a bipolar tungsten electrode (Forsythe & Barnes-Davies, 1993) at 0.05 Hz. Whole-cell voltage-clamp recordings were made using a patch-clamp amplifier (Axopatch 200B, Axon Instruments). The records were low-pass filtered at 5 kHz and digitized at 20-50 kHz by an analog-digital converter (Digidata 1322) and data were analysed with pCLAMP8.1 software (Axon Instruments). Values in the text and figures are givens as means ± S.E.M., and statistical significance was evaluated by unpaired t test, unless otherwise noted. Sample traces illustrated in figures were averaged from 8-20 events for evoked EPSCs and 240-290 events for spontaneous mEPSCs. All experiments were carried out at room temperature (22-27 °C).

Drug application

Adenosine (100 µM, Sigma/RBI), N ⁶-cyclopentyladenosine (CPA) (1 µM, Sigma/RBI), 8-cyclopentyltheophylline (CPT) (0.5 µM, Sigma/RBI), baclofen-(20 µM, Res Biochem) were dissolved in the aCSF and bath-applied by switching the superfusates. -Conotoxin GVIA (2 µM, Peptide Institute) was dissolved in aCSF containing cytochrome c (1 mg ml^-1; Sigma) just before bath application.

A₁ receptor quantification

The amount of adenosine A₁ receptor protein in the MNTB region was assessed using immunoblotting in rats at different postnatal ages. The tissues of the MNTB region were punched out from brainstem slices of two to three rats at each age, pooled and homogenized by sonication in a buffer solution containing 20 mM Tris-HCl (pH 7.4), 50 mM NaCl, 1 mM EDTA, 1 % NP-40, and 0.1 % SDS-containing protease inhibitors (Sigma). After adjusting protein concentration of all homogenates in each tube to be equal, aliquots (5 µg) from three tubes were applied to SDS-PAGE for subsequent Western blotting. Briefly, samples were (i) separated on a gradient acrylamide gel (4-20 %), (ii) electroblotted to PVDF membrane, (iii) blocked with skimmed milk, (iv) incubated with anti-adenosine A₁ receptor (Affinity Bioreagents, Golden CO, diluted 1:1000), overnight at 4 °C, (v) detected with avidin/biotin-horseradish peroxidase system according to the manufacturer's manual (Vectastain elite ABC kit, Vector laboratories) followed by ECL plus (Amersham). Chemiluminescence was measured with LAS1000 (Fuji film). To quantify the relative amount of adenosine A₁ receptor in each sample, crude homogenates of hippocampal tissue diluted into four different concentrations were simultaneously applied to the same PVDF membrane to formulate a standard curve for measuring the intensity of A₁ receptor immunoreactivity

Immunocytochemistry

Wistar rats were anaesthetized with Nembutal and transcardially superfused with a fixative (4 % paraformaldehyde and 0.2 % picric acid in 0.1 M sodium phosphate, pH 7.4). After fixation, rats were decapitated, and a tissue block of the brainstem including the MNTB region was removed for overnight postfixation at 4 °C. The fixed tissue was cryoprotected at 4 °C in 0.1 M phosphate buffer with sucrose of graded concentrations: in 4 % sucrose for 1 h, 10 % for 2 h, 15 % for 2 h, and 20 % overnight. Transverse slices (25 µm in thickness) were cut by using a cryostat (CM3050, Leica) at -21 °C. The sections were then processed for immunocytochemistry as follows: (i) blocking in 0.1 M sodium phosphate buffer containing 4 % skimmed milk and 0.4 % Triton X-100 overnight at 4 °C, (ii) application of primary antibodies in 20 mM sodium phosphate buffer solution containing 300 mM NaCl, 1 % bovine serum albumin and 0.02 % Triton X-100 for 2 days at 4 °C, (iii) application of secondary antibodies in phosphate-buffer saline solution (PBS) for 2 days at 4 °C, (iv) mounting with ProLong antifade kit (Molecular Probes). Adenosine receptors were visualized with rabbit anti-adenosine A₁ receptor antibodies (Affinity bioreagents, Golden, CO, USA, diluted 1:100). To identify the presynaptic terminal, we used a mouse anti-synaptophysin antibody (Chemicon, diluted 1:200). The secondary antibodies were goat anti-mouse IgG antibody conjugated with Alexa fluor 568 (Molecular Probes, diluted 1:200) and goat anti-rabbit IgG antibody conjugated with Alexa fluor 488 (Molecular Probes, diluted 1:200). Stained sections were viewed with a 100 oil-immersion objective (NA 1.35) using a confocal laser scanning microscope (Fluoview FV300, Olympus). Excitation wavelengths were 488 nm (argon laser) and 568 nm (krypton laser). Emission wavelengths were 510-550 nm (for green) or > 610 nm (for red). All the immunocytochemical procedures were carried out at room temperature (22-27 °C), unless noted otherwise.

	RESULTS

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Presynaptic inhibition mediated by adenosine A₁ receptors

EPSCs were recorded from MNTB principal neurons in P5-7 rats in response to stimulation of input fibres. Bath application of adenosine (100 µM) significantly attenuated the amplitude of EPSCs in all 28 cells examined, on average by 37.1 ± 2.4 % (P < 0.01), with no appreciable change in their kinetics (Fig. 1A). The inhibitory effect of adenosine was reversible after washout (Figs 1C, 3, 5, 6 and 7), but was sustained during its application (Fig. 1A), being consistent with the report that presynaptic adenosine receptors desensitize very slowly (Wetherington & Lambert, 2002). The A₁ receptor-specific antagonist CPT (0.5 µM), applied in the presence of adenosine, reversed the adenosine effect (Fig. 1A). Similar to adenosine, the A₁ receptor-specific agonist CPA (1 µM) attenuated EPSCs by 35.3 ± 9 % (n = 5), and this effect was also reversed by CPA (Fig. 1B). These results indicate that the inhibitory effect of adenosine on EPSCs is mediated by A₁ receptors. The inhibitory effect of adenosine was first detectable at 1 µM (1.4 ± 0.5 %, n = 4), and became more pronounced at higher concentrations (Fig. 1C). The concentration-response relationship for adenosine indicated an IC₅₀ of 12 µM, which is comparable to the value reported at hypothalamic synapses (Oliet & Polain, 1999).

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Figure 1. Inhibitory effect of adenosine on EPSCs mediated by A1 receptors in P5-7 rats A, bath application of adenosine (100 µM) attenuated EPSCs (b). CPT (0.5 µM) reversed this adenosine effect (c). Sample records on the top are averaged EPSCs before (a) and during (b) adenosine application, and during addition of CPT (c, superimposed). Averaged EPSCs before (a) and after (b) adenosine application, when normalized in peak amplitude, completely overlapped with each other (bottom, superimposed). B, CPA (1 µM) attenuated EPSCs (b), and this effect was reversed by CPT (0.5 µM, c). C, adenosine inhibits EPSCs in a concentration-dependent manner. Data are shown for a single cell (left) together with a concentration-inhibition curve for pooled data from four cells (right). The fitted curve derived from: y = maximum inhibition/[1 + (IC50 /adenosine concentration)nH], indicated a maximum inhibition of 40 %, an IC50 of 12 µM, and a Hill coefficient (nH) of 0.92. D, left panel, cumulative amplitude histograms of mEPSCs recorded in the presence of TTX, before and after (superimposed) application of adenosine (100 µM) showed no significant difference (Kolomogolov-Smirnov (K-S) test). Sample traces are averaged mEPSCs before and after application of adenosine (superimposed). Right panel, mean amplitude of mEPSCs before and after adenosine applications in five cells. No significant difference in paired t test. Data derived from P6 and P7 rats.

Inhibition of EPSCs by adenosine was accompanied by a significant increase in the coefficient of variation in the evoked EPSC amplitude (by 158 ± 3.4 %, n = 15, P < 0.01), as reported previously at this synapse (Barnes-Davies & Forsythe, 1995). Adenosine had no effect on the mean amplitude of spontaneous miniature (m) EPSCs recorded in the presence of tetrodotoxin (TTX, 1 µM, Fig. 1D), as reported at other synapses (Scholtz & Miller, 1992; Bagley et al. 1999; Arrigoni et al. 2001). Adenosine also had no effect on the postsynaptic holding current (1.2 ± 2.4 %, n = 10) with the Cs⁺-based internal solution in the recording pipette (see Methods). These results suggest that adenosine inhibits transmitter release by a presynaptic mechanism, as postulated at other synapses (Ginsborg & Hirst, 1972; Phillis et al. 1979; Thompson et al. 1992; Yawo & Chuhma, 1993; Umemiya & Berger, 1994; Barnes-Davies & Forsythe, 1995; Bagley et al. 1999; Arrigoni et al. 2001).

Functional role of presynaptic A₁ receptors

We next examined whether endogenous adenosine can inhibit EPSCs presynaptically. EPSCs evoked at the basal frequency (0.05 Hz) were not affected by the A₁ receptor antagonist CPT (applied for 10 min, 0.8 ± 2.2 %, n = 7, see also Fig. 2A and C), suggesting that, unlike the situation at other synapses (Arrigoni et al. 2001), ambient adenosine at the calyx of Held plays little part in regulating basal transmission. We then examined whether CPT affects EPSCs evoked at a higher frequency. During a train of stimuli at 10 Hz, EPSCs underwent a marked depression in amplitude and reached a low steady-state level (von Gersdorff et al. 1997; Takahashi et al. 2000; Iwasaki & Takahashi, 2001). Application of CPT (0.5 µM) significantly increased the steady-state EPSC amplitude during the train (P < 0.05, Fig. 2A and B) in five out of seven cells (Fig. 2C), suggesting that endogenous adenosine, elevated during high frequency synaptic activity, can inhibit EPSCs via the activation of presynaptic A₁ receptors. Thus, as previously reported at neuromuscular (Redman & Silinsky, 1994) and hypothalamic (Oliet & Poulain, 1999) synapses, adenosine derived from ATP released from nerve terminals (or postsynaptic cells) has a regulatory role in synaptic transmission. The mean magnitude of presynaptic inhibition by endogenous adenosine, deduced from the blocking effect of CPT, was 12.9 %, suggesting that during repetitive stimulation adenosine reached a concentration of nearly 5 µM at presynaptic A₁ receptors (Fig. 1C).

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Figure 2. Presynaptic inhibition by endogenous adenosine increased during high frequency stimulation A, averaged EPSCs during a train of 30 stimuli at 10 Hz. The first EPSC (I0) and the 27th-30th EPSCs, before (black) and during (red) application of CPT (0.5 µM), are shown (superimposed). B, synaptic depression during 10 Hz stimulation. EPSCs during a train are normalized in amplitude to the first EPSC. Mean amplitudes and S.E.M.s of EPSCs (from five cells with significant increase in mean amplitude of 20th- 30th EPSCs (Iss) after CPT application, D, during 10 Hz stimulation are plotted, before () and during () CPT application. C, Time plot of I0 and Iss in a cell. Bath application of CPT (bar) increased Iss with no effect on I0. Mean amplitude of Iss before CPT application is indicated by a dashed line. D, mean amplitudes of Iss before and after application of CPT in seven cells. Difference was statistically significant (*P < 0.05) in five cells, but insignificant in two other cells.

A common mechanism for the presynaptic inhibition shared by A₁ receptors and GABA_B receptors

At the calyx of Held synapse the GABA_B receptor agonist baclofen causes a strong presynaptic inhibition (Barnes-Davies & Forsythe, 1995; Takahashi et al. 1998). If A₁ receptors and GABA_B receptors share a common mechanism for presynaptic inhibition, baclofen may be expected to occlude the effect of adenosine. After measuring the control inhibitory effect of adenosine, baclofen applied at a saturating concentration (20 µM, Takahashi et al. 1998) reduced EPSCs by 87.6 ± 2.0 % (n = 5). Subsequently, in the presence of baclofen, adenosine (100 µM) no longer attenuated EPSCs (2.1 ± 1.2 %, Fig. 3c and d). After washing out baclofen, EPSCs recovered to the baseline, and a further application of adenosine reproducibly inhibited EPSCs (Fig. 3e and f). Since the EPSC amplitude remaining after baclofen application (442 ± 7.9 pA) was much greater than the noise level (2-5 pA), the lack of adenosine effect in the presence of baclofen cannot be due to the reduced signal to noise ratio. With this noise level, for example, small changes in the mean mEPSCs amplitude (~50 pA) with a large coefficient of variation (~0.5) can be reliably measured (Yamashita et al. 2003). Thus these results of the occlusion test strongly suggest that A₁ receptors and GABA_B receptors share a common mechanism.

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Figure 3. Baclofen occludes the inhibitory effect of adenosine In the presence of baclofen (20 µM), the inhibitory effect of adenosine (100 µM) on EPSCs (a, b) was abolished (c, d). After baclofen washout, the adenosine effect was recovered (e, f). Sample traces are averaged EPSCs before and after adenosine application (superimposed) in the absence of baclofen (a, b), in the presence of baclofen (c, d), and after baclofen washout (e, f). The lower panel shows EPSC amplitude data for a single cell during 100 min of recording.

To clarify the target of adenosine-induced presynaptic inhibition, we examined the effect of adenosine on the presynaptic Ca²⁺ currents (I_pCa) recorded directly from the calyceal terminal (Takahashi et al. 1996, 1998). Under the whole-cell voltage-clamp recording, I_pCa was evoked by a depolarizing pulse stepping from a holding potential of -80 mV to 0 mV (Fig. 4A). Bath application of adenosine (100 µM) clearly inhibited I_pCa (Fig. 4A) in a reversible manner (data not shown, see Fig. 5). The magnitude of inhibition of I_pCa by adenosine was 20.3 ± 4.4 % (n =5).

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Figure 4. Inhibitory effect of adenosine on presynaptic Ca2+ currents A, Ca2+ currents were evoked by a depolarizing command pulse (10 ms) stepping from -80 mV to 0 mV (a). Adenosine (100 µM) attenuated Ca2+ currents (b). Current-voltage relationships before () and after () adenosine application (from 5 cells in P6-7 rats). Current amplitudes were measured 2 ms after the onset of command pulse and normalized to the control value at 0 mV. B, lack of effect of adenosine on presynaptic voltage-dependent potassium currents. Sample traces in inset are outward potassium currents evoked by 20 mV depolarizing steps from the holding potential of -70 mV to +30 mV in the presence of TTX before (left) and after (middle) adenosine application. The superimposed traces before and after adenosine application overlap almost completely (right). In the current-voltage relationship, the amplitudes of potassium currents were normalized to the control value at +10 mV. Mean values ± 5 S.E.M.s of 6 calyces before () and after () adenosine applications are plotted against membrane potential.

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Figure 5. Comparison of IpCa-EPSC relationships during adenosine application and [Ca2+]o reduction Paired recording from a calyx and its target MNTB neuron. A, EPSCs were evoked by a depolarizing pulse (1 ms duration) stepping from -70 mV to +40 mV. In the lower panels, the application of adenosine (100 µM) is indicated by a bar, and the three switches to an external solution with low [Ca2+]o/[Mg2+]o (0.5 mM/3.0 mM; each for 10-30 s) are indicated by arrowheads. IpCa (pre) and EPSC (post) before (black) and during applications of adenosine or low [Ca2+]o solution (red) are superimposed. B, the IpCa-EPSC relationship in double logarithmic plots, with data points and regression lines during adenosine application (red) and [Ca2+]o/[Mg2+]o reductions (black). Regression lines were drawn according to the least square method. C, the slope values of the regression lines compared between adenosine and low [Ca2+]o/[Mg2+]o at seven synapses. No significant difference (P = 0.81) in paired t test. The mean slope values were 1.74 ± 0.27 for adenosine and 1.75 ± 0.25 for [Ca2+]o/[Mg2+]o reduction, respectively.

We next examined whether adenosine might affect K⁺ channels. Voltage-dependent outward K⁺ currents were evoked by depolarizing presynaptic terminals in the presence of TTX (1 µM) (Forsythe, 1994; Ishikawa & Takahashi, 2001). As shown in Fig. 4B, adenosine had no effect on the K⁺ currents. Adenosine also had no effect on the holding current (1.7 ± 3.9 %, n = 6), suggesting that adenosine does not affect the G protein-coupled inward rectifying potassium conductance found at the calyx of Held (Takahashi et al. 1998).

We next examined whether the exocytotic machinery downstream of Ca²⁺ influx might also be involved in A₁ receptor-mediated presynaptic inhibition. In simultaneous pre- and postsynaptic recordings, EPSCs were evoked by I_pCa elicited by a brief (0.5-1 ms) depolarizing pulse. Application of adenosine or a reduction of the [Ca²⁺]_o/[Mg²⁺]_o ratio attenuated both I_pCa nd EPSCs in parallel (Fig. 5A). When the I_pCa-EPSC relationships were plotted in double logarithmic co-ordinates (Fig. 5B) the relationships during adenosine application and during [Ca²⁺]_o/[Mg²⁺]_o reduction largely overlapped with each other. At seven synapses examined, the slope of the regression line after adenosine application was similar to that after reduction of the [Ca²⁺]_o/[Mg²⁺]_o ratio (Fig. 5C, no significant difference in paired t test). Thus, the adenosine-induced inhibition of I_pCa fully explains the reduction of the EPSC amplitude.

Developmental decline in the magnitude of presynaptic inhibition by adenosine

The inhibitory effect of adenosine on EPSCs was robust at P5-7, but markedly diminished with development (Fig. 6). At P14-16 or P20-21, adenosine-induced inhibition of EPSCs was still observed in all cells examined (10.4 ± 3.1 %, n = 5 at P14-16; 11.6 ± 3.6 %, n = 5 at P20-21), but the magnitudes of inhibition were significantly less than that at P5-7 (37.1 ± 2.4 %, n = 28, P < 0.01, ANOVA). At P14-16 CPT no longer relieved synaptic depression during stimulation at 10 Hz (data not shown).

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Figure 6. Developmental decline in adenosine-induced presynaptic inhibition A, adenosine (100 µM) only slightly attenuated EPSCs at P14 and P21. Sample traces are averaged EPSCs before (a) and after (b) adenosine applications (superimposed) in P14 and P21 rats. B, the magnitude of inhibition of EPSCs by adenosine at different postnatal periods; inhibition at P6-7 is significantly larger than at P14-16 or P20-21 (* P < 0.01, ANOVA).

During the second postnatal week, at the calyx of Held terminal, Ca²⁺ channels triggering transmitter release switch from a mixture of N-, P/Q- and R-types to a single P/Q-type (Iwasaki & Takahashi, 1998; Iwasaki et al. 2000). A₁ receptors can be selectively linked to N-type Ca²⁺ channels (Yawo & Chuhma, 1993; Umemiya & Berger 1994), suggesting that the developmental decline in the effect of adenosine at the calyx of Held synapse might arise from the developmental decline of N-type Ca²⁺ channels. To test this possibility we compared the effect of adenosine before and after application of the N-type-specific Ca²⁺ channel blocker -conotoxin GVIA (-CgTX, 2 µM) in P5-7 rats. Whereas -CgTX decreased EPSCs (Fig. 7) by 23.3 ± 3.6 % (n = 4) as reported (Iwasaki & Takahashi, 1998; Wu et al. 1999), it did not change the magnitude of adenosine-induced inhibition of EPSCs (Fig. 7B). The magnitudes of adenosine-induced EPSC inhibition before (38.8 ± 5.1 %) and after -CgTX application (40.2 ± 5.8 %) in four cells were not significantly different (P = 0.54, paired t test). These results indicate that adenosine attenuates N-type Ca²⁺ channels and other Ca²⁺ channels (P/Q-type and R-type channels, Iwasaki & Takahashi, 1998; Wu et al., 1999) to a similar extent. Thus the developmental decline in the adenosine-induced presynaptic inhibition cannot result from the developmental switch of Ca²⁺ channel subtypes.

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Figure 7. -CgTX GVIA had no effect on the magnitude of adenosine-induced inhibition of EPSCs A, -CgTX (2 µM) attenuated EPSCs (c), but did not affect the magnitude of adenosine-induced inhibition of EPSCs. Sample records are averaged EPSCs before and after adenosine application before (a, b, superimposed, left) and after -CgTX application (c, d, right). B, the magnitude of adenosine-induced EPSC inhibition before and after -CgTX application in four cells. No significant difference (P = 0.54) in paired t test.

To examine whether the expression of A₁ receptors decreases with development, we carried out Western blot analysis for the A₁ receptor protein in the MNTB region. As shown in Fig. 8, the immunoreactive bands are found at the position expected from the molecular mass of the A₁ receptor. The amount of A₁ receptor protein, deduced from the intensity of immunoreactivity, diminished with development from P7 to P14, and reached a steady level thereafter. Although A₁ receptor proteins thus measured derived from both presynaptic and postsynaptic sites, the time course of decline was similar to that of adenosine-induced EPSC inhibition (Fig. 6).

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Figure 8. Western blot analysis of A1 receptors Samples from the MNTB region at each age (P7, P16 and P21) were separated with SDS-PAGE for immunoblot detection. Arrowheads indicate the positions corresponding to the molecular mass, and an open arrowhead indicates the position expected for A1 receptors. The A1 receptor signal intensity at each lane was measured using densitometry, and normalized to the mean value (of three samples, two of which are shown) at P7. Bar graphs indicate the mean values and S.E.M. (from three experiments) for the A1 receptor signal intensity representing the relative amount of A1 receptor protein, which was significantly different between P7 and P16, and between P7 and P21 (* P < 0.01, ANOVA).

The large nerve terminal of the calyx of Held synapse is advantageous for the immunocytochemical identification of various presynaptic proteins (Kajikawa et al. 2001; Saitoh et al. 2001). At P7 diffuse A₁ receptor immunoreactivity (green) was found in the MNTB neuron, and at the presynaptic terminal as identified with an overlap with the immunoreactivity of synaptophysin (red, Fig. 9). These signals were significantly stronger than the background immunofluorescence signal for the secondary antibody (Fig. 9A). Given that the confocal microscopy has a z-axis half-width at half-maximum (HWHM) of about 340 nm and x- and y-axis resolutions of about 200 nm, it can sufficiently identify a calyceal terminal having a diameter of 1-2 µm. The laser scanning also minimizes the fluorescence blur. For example, even a strong immunofluorescence signal for the postsynaptic marker MAP2B does not overlap with the presynaptic signal of syntaxin at the calyx of Held (Tsujimoto et al. 2002). Thus, the intensity of A₁ receptor immunofluorescence in the overlapped region (yellow) represents the level of presynaptic A₁ receptor immunoreactivity. From P7 to P14, A₁ receptor immunoreactivity at the nerve terminal was reduced by 26.8 % (Fig. 9B). The presynaptic A₁ receptor immunoreactivity then remained at a similar level until P21 (Fig. 9B). Similarly the A₁ receptor immunoreactivity in the MNTB cell body decreased from P7 to P14 and reached a low level (23.8 % at P14, 22.1 % at P21). These results suggest that the expression of A₁ receptors at the calyx of Held presynaptic terminal declines during the second postnatal week.

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Figure 9. Developmental reduction in the immunoreactivity of A1 receptors at the calyx of Held synapse A, A1 receptor immunoreactivity is visualized with Alexa fluor 488 (green). As a presynaptic marker, synaptophysin immunoreactivity was visualized with Alexa fluor 568 (red). The presynaptic intensity of A1 receptor immunofluorescence deduced from an overlap with synaptophysin immunofluorescence (yellow) decreased from P7 to P14. Postsynaptic A1 receptor immunofluorescence (green, not overlapped with synaptophysin) similarly decreased in intensity with development. Bottom left, background signal of secondary antibody (A1R (-)). B, densitometric quantification of the presynaptic A1 receptor immunofluorescence. Bar graphs represent the mean A1 receptor signal intensity at each age relative to the mean value at P7. Error bars indicate +S.E.M. of 7-11 cells (numbers indicated in each column). * Significant difference between P6-7 and P14-16, and between P6-7 and P20-21 (P < 0.01, ANOVA).

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

A common intracellular mechanism shared by multiple presynaptic receptors

At the calyx of Held synapse, adenosine inhibited EPSCs via activation of presynaptic A₁ receptors. Adenosine also inhibited presynaptic Ca²⁺ currents without affecting K⁺ currents. The magnitude of inhibition of presynaptic Ca²⁺ currents by adenosine almost fully explained its presynaptic inhibitory effect on the evoked EPSCs, as is the case for L-AP4 (Takahashi et al. 1996) and baclofen (Takahashi et al. 1998). Baclofen at its maximal concentration (Takahashi et al. 1998) occluded the presynaptic inhibitory effect of adenosine (Fig. 3) as well as that of L-AP4 (Yoshinao Kajikawa and Tomoyuki Takahashi, unpublished observation). These results suggest that A₁ receptors, mGluRs and GABA_B receptors in the calyx of Held terminal share a common mechanism for the presynaptic inhibition. At this nerve terminal baclofen activates G protein Go, and its subunits inhibit Ca²⁺ channels via a membrane-delimited pathway (Kajikawa et al. 2001). This intracellular signal pathway is likely to be shared by A₁ receptors, mGluRs and GABA_B receptors. It has recently been reported that noradrenaline, via activating ₂-adrenergic receptors, inhibits presynaptic Ca²⁺ currents (Leao & von Gersdorff, 2002). Thus ₂-adrenergic receptors may also share the same mechanism. Whereas multiple types of GPCRs mediate presynaptic inhibition at central synapses, our results suggest that multiple GPCRs are linked to a common target at a single nerve terminal for the regulation of transmitter release.

The N-type Ca²⁺ channel blocker -CgTX GVIA can abolish the presynaptic inhibitory effect of adenosine (Yawo & Chuhma, 1993; Umemiya & Berger 1994) and the A₁ receptor-mediated Ca²⁺ current inhibition in neuronal cell somata (Mogul et al. 1993). -CgTX can also abolish the presynaptic inhibitory effect of dopamine (Momiyama & Koga, 2001). In cultured secretory cells (Currie & Fox, 1997) and Xenopus oocytes (Zhang et al. 1996), G protein activators preferentially inhibit N-type Ca²⁺ channels and have much less effect on P/Q-type Ca²⁺ channels. Together these reports suggest that GPCRs are tightly linked to N-type Ca²⁺ channels. However, at the calyx of Held synapse in P6-7 rats, the magnitude of adenosine-induced EPSC inhibition did not change even after EPSCs were attenuated by -CgTX application, indicating no selective linkage between A₁ receptors and N-type Ca²⁺ channels at this synapse. Thus the apparently selective linkage between GPCRs and N-type Ca²⁺ channels at other synapses might reflect their closer localization in the nerve terminal relative to other type of Ca²⁺ channels. Another possibility would be that a splice variant of the N-type Ca²⁺ channel _1B subunit (Williams et al. 1992) might selectively couple with GPCRs.

Physiological role of presynaptic A₁ receptors

At the calyx of Held synapse in P5-7 rats, blocking A₁ receptors with CPT significantly relieved synaptic depression (Fig. 2), suggesting that endogenous adenosine contributes to synaptic depression. Adenosine derives from ATP co-released from the calyx of Held terminal with the transmitter glutamate and/or from surrounding MNTB cells activated by synaptic inputs. Similar to adenosine, glutamate released from the calyceal nerve terminal contributes to synaptic depression by activating presynaptic mGluRs (von Gersdorff et al. 1997; Iwasaki & Takahashi, 2001). Thus, at the immature calyx of Held synapse, both mGluRs and A₁ autoreceptors regulate transmitter release during high frequency transmission, thereby limiting transmitter depletion. This role seems particularly important during the immature period at which synaptic vesicles can be depleted more easily during repetitive transmission because of a relatively high release probability (Taschenberger & von Gersdorff, 2000; Iwasaki & Takahashi, 2001).

In various brain regions, the concentration of ambient adenosine is estimated to be 1-2 µM (Latini & Pedata, 2001). Inhibition of EPSCs by 1 µM adenosine was just detectable at the calyx of Held synapse (Fig. 1C). In the present experiments, however, CPT had no appreciable effect on EPSCs evoked at a basal frequency (0.05 Hz), suggesting that extracellular ambient adenosine concentration was less than 1 µM in our slices. Ambient adenosine concentration may be higher in vivo, where the neuronal activity is higher, and it will rise during hypoxia (Latini & Pedata, 2001). Given the remarkably slow desensitization of presynaptic A₁ receptors (Wetherington & Lambert, 2002), ambient adenosine will provide a sustained inhibition of transmitter release, thereby preserving transmitter glutamate, and protecting cells from possible glutamate toxicity.

Developmental change in presynaptic receptors

At the calyx of Held synapse, during the second postnatal week, A₁ receptor expression declines (Fig. 9) in parallel with the adenosine-induced presynaptic inhibition (Fig. 6). In contrast the presynaptic inhibitory effect of baclofen remains similar during the second postnatal week (Takahashi et al. 1998). The presynaptic inhibitory effect of L-AP4 also remains similar during this period (Takahashi et al. 1996; Iwasaki & Takahashi, 2001). However, contribution of mGluRs to synaptic depression becomes undetectable at P14, suggesting that the glutamate concentration reaching presynaptic mGluRs is reduced with development possibly because of an increased glutamate uptake (Iwasaki & Takahashi, 2001). During the second postnatal week, the magnitude of noradrenaline-induced presynaptic inhibition does not change, but the percentage of synapses responsive to noradrenaline decreases (Leao & von Gersdorff, 2002). Thus developmental changes in the presynaptic receptors at the calyx of Held are diverse, but directed toward a decrease in their contribution to the regulation of transmitter release (except for GABA_B receptors). Similarly at thalamocotical synapses, presynaptic inhibition by kainate autoreceptors declines during the first postnatal week (Kidd et al. 2002). At hippocampal CA1 synapses, mGluR-mediated presynaptic inhibition decreases after one postnatal month (Baskys & Malenka, 1991). Thus, at many central synapses, regulation of transmitter release by presynaptic GPCRs peaks during the early postnatal period. It remains to be seen what kind of signal cascade underlies these developmental changes.

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Acknowledgements

This study was supported by Grant-in-Aid for Specially Promoted Research from the Ministry of Education, Culture, Sports, Science and Technology. We thank Yoshinao Kajikawa for technical advice. We also thank Mark Farrant, Taro Ishikawa, Shinichi Iwasaki and Tetsuhiro Tsujimoto for helpful comments on the manuscript.

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