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¹ Department of Cellular Neuroscience, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan
² Division of Cell Biology, Department of Molecular and Cellular Biology, Kobe University Graduate School of Medicine, Kobe, Hyogo 650-0017, Japan
³ Division of Neurophysiology, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan
⁴ Department of Physiology and Pharmacology, Karolinska Institutet, S-171 77 Stockholm, Sweden
⁵ Department of Neurobiology and Behaviour, Gunma University Graduate School of, Medicine, Maebashi, Gunma 371-8511, Japan

	Abstract

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Adenosine receptors (ARs) are G protein-coupled receptors (GPCRs) mediating the neuromodulatory actions of adenosine that influence emotional, cognitive, motor, and other functions in the central nervous system (CNS). Previous studies show complex formation between ARs and metabotropic glutamate receptors (mGluRs) in heterologous systems and close colocalization of ARs and mGluRs in several central neurons. Here we explored the possibility of intimate functional interplay between G_i/o protein-coupled A₁-subtype AR (A1R) and type-1 mGluR (mGluR1) naturally occurring in cerebellar Purkinje cells. Using a perforated-patch voltage-clamp technique, we found that both synthetic and endogenous agonists for A1R induced continuous depression of a mGluR1-coupled inward current. A1R agonists also depressed mGluR1-coupled intracellular Ca²⁺ mobilization monitored by fluorometry. A1R indeed mediated this depression because genetic depletion of A1R abolished it. Surprisingly, A1R agonist-induced depression persisted after blockade of G_i/o protein. The depression appeared to involve neither the cAMP-protein kinase A cascade downstream of the alpha subunits of G_i/o and G_s proteins, nor cytoplasmic Ca²⁺ that is suggested to be regulated by the beta-gamma subunit complex of G_i/o protein. Moreover, A1R did not appear to affect G_q protein which mediates the mGluR1-coupled responses. These findings suggest that A1R modulates mGluR1 signalling without the aid of the major G proteins. In this respect, the A1R-mediated depression of mGluR1 signalling shown here is clearly distinguished from the A1R-mediated neuronal responses described so far. These findings demonstrate a novel neuromodulatory action of adenosine in central neurons.

(Received 5 February 2007; accepted after revision 16 March 2007; first published online 22 March 2007)
Corresponding author M. Kano: Department of Cellular Neuroscience, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan. Email: mkano{at}cns.med.osaka-u.ac.jp

	Introduction

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Adenosine is a ubiquitous neuromodulator in the mammalian CNS. Adenosine is derived from neurons and glia, and accumulates in the extracellular fluid (Fredholm et al. 2001; Ribeiro et al. 2003). Adenosine activates widely distributed G protein-coupled receptors (GPCRs) named A₁, A_2A, A_2B, and A₃ receptors (A1R, A2AR, A2B, and A3R, respectively; collectively, ARs). ARs regulate arousal level and motor activity, prevent anxiety-related behaviour and epileptiform discharges, and affect neurodegeneration, cognition, and learning (Rudolphi et al. 1992; Schubert et al. 1997; Nyce, 1999; Fredholm et al. 2000; Haas & Selbach, 2000; Dunwiddie & Masino, 2001; Johansson et al. 2001; Ribeiro et al. 2003).

Inhibition of excitatory synaptic transmission is thought to be a key process for these effects of adenosine (Dunwiddie & Masino, 2001). The best studied are the inhibition of synaptic glutamate release by A1R and the inhibition of N-methyl-D-aspartate receptor conductance by A2AR (Haas & Selbach, 2000; Wirkner et al. 2000; Dunwiddie & Masino, 2001). Both types of inhibition attenuate ionotropic glutamate receptor signalling in the postsynaptic neurons. It remains unclear whether and how adenosine in addition influences metabotropic glutamate receptor (mGluR) signalling, which plays important roles in induction of slow excitatory postsynaptic potentials (EPSPs) (Batchlor & Garthwaite, 1997; Tempia et al. 2001), intracellular Ca²⁺ mobilization (Lliano et al. 1991; Finch & Augustine, 1998; Takechi et al. 1998), synaptic plasticity (Aiba et al. 1994; Conquet et al. 1994; Shigemoto et al. 1994; Ichise et al. 2000), production of endocannabinoids (Maejima et al. 2001; Maejima et al. 2005), and developmental synapse elimination (Kano et al. 1997; Ichise et al. 2000). Some studies have revealed that A1R and A2AR can form complexes with group-I mGluRs in non-neuronal heterologous expression systems and that immunoreactivities for A1R and mGluR1 overlap closely in several central neurons including cerebellar Purkinje cells (Ciruela et al. 2001; Ferre et al. 2002). These observations suggest the possibility of intimate functional interplay between neuronal ARs and mGluRs.

In this study, we explored possible functional interplay from native A1R to native mGluR1 in cerebellar Purkinje cells (Houamed et al. 1991; Masu et al. 1991; Reppert et al. 1991; Svenningsson et al. 1997; Ciruela et al. 2001). In cerebellar slice preparations, it was difficult to distinguish AR–mGluR1 interplay in Purkinje cells from synaptic modulation mediated by presynaptic ARs (Dittman & Regeher, 1996; our unpublished data). Therefore, we used isolated Purkinje cell preparations. We monitored mGluR1 signalling, using two types of G_q protein-mediated responses: an inward cation current through transient receptor potential C1 subunit-containing channels (Kim et al. 2003; Hartmann et al. 2004) and Ca²⁺ release from inositol trisphosphate receptor (IP₃R)-equipped intracellular stores (Lliano et al. 1991; Finch & Augustine, 1998; Takechi et al. 1998; Miyata et al. 2000). We have found that A1R agonists depress both types of mGluR1-coupled responses. We have confirmed using A1R-knockout (A1R-KO) mice (Johansson et al. 2001) that A1R indeed mediates this depression. Surprisingly, the depression does not require G_i/o proteins, unlike the classical A1R-mediated neuronal responses. These findings demonstrate a novel neuromodulatory action of adenosine in central neurons.

	Methods

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Cell culture

Cerebellar Purkinje cells from wild-type C57BL/6 mice were cultured as described elsewhere (Tabata et al. 2000). Briefly, perinatal embryos were caesarean-sectioned from pregnant mice deeply anaesthetized and killed with diethylether or isoflurane. The embryos were deeply anaesthetized by cooling in chilled phosphate-buffered saline and then killed by decapitation. The cerebella from these embryos were dissociated with trypsin and plated onto plastic dishes (diameter 35 mm; Falcon 3001, Becton Dickinson, Franklin Lakes, NJ, USA) or low-fluorescence plastic films (Sumilon MS-92132, Sumitomo, Tokyo, Japan), and maintained for 11 days to 3 weeks in a medium based on 1 : 1 mixture of Dulbecco's modified Eagle medium and F-12 nutrients (DMF; Gibco 12400, Life Technologies, Grand Island, NY, USA). In some experiments, cerebellar neurons were dissected from newborn pups generated by mating the homozygous A1R-KO (A1R(–/–)) mice (Johansson et al. 2001) that were backcrossed to C57BL/6 strain according to Jackson Laboratories' specified congenic procedure. Purkinje cells were identified by their large somata and thick primary dendrites (Tabata et al. 2002). The experiments in this study are approved by the committees on animal experiments of Kanazawa University and Osaka University, and the procedures mentioned above fully conform to the guidelines administered by these committees.

Electrophysiology

Somatic whole-cell recordings were made from Purkinje cells cultured on the dish in a perforated-patch (Horn & Marty, 1988) or ruptured-patch mode (Marty & Neher, 1995). The pipette solution used in perforated-patch recordings consisted of (mM): 95 Cs₂SO₄, 15 CsCl, 0.4 CsOH, 8 MgCl₂, 10 Hepes, and 200 µg ml^–1 amphotericin B (pH 7.35). The pipette solution used in a ruptured-patch recordings consisted of (mM): 130 K-D-gluconate, 10 NaCl, 10 Hepes, 0.5 ethylene glycolbis(-aminoethylether)N,N,N',N'-tetraacetic acid, 4 Mg-ATP, 0.4 Na₂-GTP; the total Mg²⁺ level was adjusted to 5.2 mM with MgCl₂; the total K⁺ level and pH were adjusted to 150.6 mM and 7.3, respectively, with KCl, KOH, and/or D-gluconic acid. The recording chamber (culture dish) was perfused at a rate of 2 ml min^–1 (0.7–1 ml min^–1 for experiments in Fig. 8) with a saline solution. The standard saline consisted of (mM): 116 NaCl, 5.4 KCl, 1.1 NaH₂PO₄, 23.8 NaHCO₃, 2.3 MgCl₂, 5.5 D-glucose, and 5 Hepes (pH 7.3, 25°C). Voltage-gated Na⁺ channels and ionotropic receptors for glutamate and GABA were blocked by supplementing the saline with the following antagonists unless otherwise stated (µM): 0.3 tetrodotoxin, 10 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfomide, 50 D(–)-2-amino-5-phosphonopentanoic acid or 10 3-((R)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid, and 10 (–)-bicuculline methochloride. Command potentials were corrected for a liquid junction potential between the pipette and bath solutions. Signals were low-pass-filtered at 0.05–2 kHz and sampled at 0.1–10 kHz, using a voltage-clamp amplifier (EPC 9/2, HEKA, Lambreht, Germany) controlled by PULSE software (version 8.53, HEKA). We measured mGluR1-coupled and ionotropic glutamate receptor-mediated currents and the direct effect of an A1R agonist in the perforated-patch mode (series resistance (R_series) 50 M) and G_i/o protein-coupled inwardly rectifying K⁺ currents in the ruptured-patch mode (R_series, 15 M; electronical compensation, 60%). For depicting the ramp I–V relation of the inwardly rectifying currents, the membrane potential was swept from –31 mV to –131 mV for 1 s in the saline supplemented with 10.6 mM KCl; the E_K estimated from the Nernst equation was –57.6 mV. We recorded the hyperpolarization-activated mixed cation current (I_h) in the ruptured-patch mode (R_series, 15 M; electronic compensation, 60%). In this experiment, MgCl₂ in the perfusate was replaced partially with 1 mM BaCl₂ and 1 mM CaCl₂ to minimize inwardly rectifying K⁺ currents (Tabata et al. 2005).

View larger version (22K): [in this window] [in a new window]   Figure 8.  A1R-mediated mGluR1 depression is not associated with a change in the background conductance A, sample voltage step-evoked currents of a Purkinje cell recorded before (Basal) and during local application of R-PIA (50 nM). Each trace indicates the average of three records obtained at the labelled time after R-PIA onset. Dotted line, zero-current level. The input resistance (Rinput) was estimated from a difference between mean current levels over the last 95 ms of ±5 mV steps. Holding potential, –70 mV (perforated-patch mode with the Cs+-containing pipette solution). B and C, the mean holding current levels (Ihold) (average level over a 95 ms period prior to the first voltage step; an inward deflection indicated as a positive value) and mean Rinput at the labelled period. For each cell, the basal value was taken as 100%. n = 5 cells. NS, P > 0.05 compared with the basal values (paired t test for the raw data). D, sample voltage step (±5 mV)-evoked currents before (Basal) and during (at 12–13 min after drug onset, Test) local application of CCPA (500 nM), 2-CA (30 µM), or adenosine (400 nM). Each pair of traces was obtained from a different cell, using the same procedures as in A.

Purkinje cells cultured on the film were loaded with fura-2, a Ca²⁺ indicator by incubation with fura-2 acetoxymethyl ester (5 µM, 37°C, 15 min). Then, the film with the cells was placed on a glass-based recording chamber and perfused at a rate of 1–1.5 ml min^–1 with a saline solution (see above); 2.3 mM MgCl₂ was partially replaced with 2 mM CaCl₂. [Ca²⁺]_i-dependent fluorescence signals were captured at 2–5 Hz, using an imaging system (Polychrome II, TILL, Planegg, Germany) attached to an inverted microscope (NA = 0.75; IX70, Olympus, Tokyo, Japan). A change in the [Ca²⁺]_i was expressed as a change in the ratio of somatic fluorescence signals excited at 340 and 380 nm (exposure duration, 40 and 20 ms, respectively) (change in F₃₄₀/F₃₈₀).

Drug application

Test drugs were dissolved into water to a concentration 200–1000 times higher than the final level (stock solution) and kept at –20°C or 4°C until use. The stock solutions of (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazoleprop-ionic acid (AMPA) and KT5720 were prepared with 10 mM Hepes buffer (pH adjusted to 7.1 with NaOH) and DMSO, respectively, and kept at –20°C until use. Adenosine was dissolved directly into the saline within 1 h before use.

Local application of agonists for mGluR1 and A1R, AMPA, and KT5720 was done by delivering saline containing each agent through a single-barrel glass pipette (i.d., 0.86 mm; o.d., 1.5 mm) or one of the two barrels of a theta glass pipette (outer diameter, 1.5 mm; BT150-10, Sutter, Novato, CA, USA). The diameter of a barrel opening was 150–200 µm. The pipette tip was located so that direct fluid stream encompassed the dendrites and soma of the examined cell (distance from the tip to the cell, 1–1.2 mm). Fluid flow was driven by gravity (gap between the heights of the reservoir and the pipette tip, 20–40 cm). The timing of a short-term local application was regulated by an electromagnetic valve (VM8, ALA, Westbury, NY, USA) controlled by the imaging system or Master-8 (AMPI, Jerusalem, Israel). For bath application, the recording chamber was perfused with saline containing AR agonists (2 ml min^–1), baclofen (2 ml min^–1), or NF023 (1 ml min^–1).

In some experiments, Purkinje cells were pretreated with pertussis toxin (PTX) by adding DFM containing 50 µg ml^–1 PTX with or without 1% v/v bovine serum albumin to the culture medium at 1 : 100.

Data analysis

To measure the peak amplitudes of mGluR1-coupled and AMPA-evoked currents, the current records were further filtered at 1 Hz and 30 Hz off line, respectively. The data of mGluR1-coupled [Ca²⁺]_i rises were discarded when there was a drastic change in the kinetics between the basal and test records. The peak amplitude was measured as a difference from the prestimulus level to the maximal deflection throughout a 10 s agonist application (for both mGluR1-coupled responses) or throughout the record (for the AMPA-evoked current). The resting [Ca²⁺]_i is presented by the mean level over 4 s prior to agonist application. The steady-state amplitude of the time-dependent component of I_h was measured as a difference from the level upon a test potential-evoked capacitive transient to the level at the end of the test potential step. A numerical data group is presented as mean ± S.E.M. throughout the text and figures. Differences between raw-value data groups were examined by ANOVA and unpaired t test unless otherwise stated. Differences between percentage-scored data groups were examined by rank score tests because the original distribution of the values could be distorted after scoring. When both percentage-scored data groups to be compared were judged to have normal distribution (P > 0.05, Shapiro–Wilk W test), the Van der Waerden test was used; otherwise, the Wilcoxon (Mann–Whiteny U) test was used.

	Results

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A1R activation depresses mGluR1 signalling

First, we explored the effects of AR subtype-specific agonists on mGluR1 signalling in cultured cerebellar Purkinje cells. To ensure activation of a majority of the target receptors, we applied the corresponding agonist at a dose at least 10 times higher than the reported binding affinity (Abbracchio et al. 1997; Poulsen & Quinn, 1998; Klotz, 2000; Fredholm et al. 2001). We evaluated the intensity of mGluR1 signalling by the peak amplitude of a R,S-3,5-dihydroxyphenylglycine (DHPG, a group-I mGluR-selective agonist, 50 µM)-evoked inward cation current that is coupled to mGluR1 exclusively via G_q protein (Hartmann et al. 2004) (Figs 1–7 and 10). We have previously reported that extracellular Ca²⁺ increases the ligand sensitivity of mGluR1 via GABA_BR (Tabata et al. 2002; Tabata et al. 2004). To exclude this effect of extracellular Ca²⁺, we used Ca²⁺-free saline unless otherwise stated. The peak latency of the inward current ranged typically between 2 s and 8 s (Figs 1–7 and 10). This deviation was not due to a change in drug application speed because a current evoked by AMPA delivered through the same local applicator (see below, cf. Figure 9A) did not show such a large deviation in activation kinetics (10–90% rise time, 137.2 ± 15.0 ms, n = 5). Despite the intrinsic deviation in the kinetics, the peak amplitude of the DHPG-evoked inward current is a reliable index of mGluR1 activity because there is only a small cell-to-cell deviation in the DHPG dose dependence of the peak amplitude (Tabata et al. 2002; Tabata et al. 2004).

View larger version (20K): [in this window] [in a new window]   Figure 1.  A1R activation depresses mGluR1 signalling A–C, bath application of A1R-selective agonists (R-PIA, 50 nM, 7 cells; CCPA, 500 nM, 7 cells) but not the normal saline (control, 7–8 cells) depresses mGluR1-coupled inward currents evoked by DHPG (50 µM, applied locally for 10 s) in cerebellar Purkinje cells. The cells were voltage-clamped at –70 mV in a perforated-patch whole-cell mode. Traces in each panel indicate sample responses recorded from a single cell before (Basal), during (Test), and after (Recov.) bath application. Arrowhead, DHPG onset. Each plot summarizes the mean peak amplitude of the inward currents as a function of time. For each cell, the peak amplitude is percentage scored, taking the average of three basal records as 100%. Thick bar, bath application period. The same conventions of data acquisition and presentation apply to Figs 1–6 and 10. In some cases with R-PIA, bath application of this agent was extended to seek the plateau of its effect (see Results); the commencement of the recovery time courses was aligned to a time of 12 min in the plot. D, comparison of the effects of the labelled test agents by the mean peak amplitudes at 12 min of bath application onset. Data were collected from the same cells used in A–C. *P < 0.05 and **P < 0.01 compared with the control, respectively (rank score test).

View larger version (11K): [in this window] [in a new window]   Figure 7.  Gq protein is not coupled to A1R A, local application of R-PIA (500 nM, 30) as well as the normal saline (30 s) does not evoke an inward current, unlike DHPG (50 µM, 10 s). Traces indicate the sample responses of a cell. Arrowheads, local application onset. Holding potential, –70 mV (perforated-patch whole-cell mode). The GIRK current was blocked with 5 mM Cs+ in the saline (Tabata et al. 2005). B, comparison of the mean maximal inward deflections during application of the labelled test drugs. NS, P > 0.05 and **P < 0.01 compared with the control, respectively (ANOVA and paired t test).

View larger version (21K): [in this window] [in a new window]   Figure 10.  Physiological levels of adenosine depress mGluR1 signalling A and B, bath application of an endogenous A1R agonist (adenosine) at physiological concentrations (40 nM, 8 cells; 400 nM, 7 cells) depresses DHPG (50 µM, 10 s)-evoked inward currents. C, comparison of the effects of the labelled test agents by the mean peak amplitudes at 12 min after bath application onset. Control, the normal saline (8 cells, reproduced from Fig. 1). 40 nM and 400 nM, data with adenosine collected from the same cells used in A and B, respectively. *P < 0.05 compared with the control (rank score test).

View larger version (21K): [in this window] [in a new window]   Figure 9.  A1R-mediated depression is not observed for AMPAR-mediated signalling A, sample AMPA (10 µM)-evoked currents of a Purkinje cell recorded before (Basal) and during bath application of R-PIA (50 nM). Each trace indicates the average of three records obtained at the labelled time after R-PIA onset. AMPA-containing saline was applied for 100 ms locally through one of the two barrels of a theta pipette, and then the control saline was applied for 1 s through the other barrel to wash out AMPA. Holding potential, –70 mV (perforated-patch mode with the Cs+-containing pipette solution and the standard saline without a blocker against AMPA-type glutamate receptors). B, the mean peak amplitudes of the AMPA-evoked currents at the labelled period. For each cell, the basal value was taken as 100%. Data obtained from 5 cells. NS, P > 0.05 compared with the basal values (paired t test for the raw data).

CGS21680 (150 nM, n = 7), an A2AR-selective agonist and 2-(1-hexynyl)-N-methyladenosine (HEMADO, 30 nM, n = 7), an A3R-selective agonist did not clearly depress the inward current (Figs 2A and B). The minimal peak amplitudes measured with CGS21680 (89.5 ± 8.3% of the basal level, at 3 min after application onset) and HEMADO (85.7 ± 5.6%) were not smaller than that of the control (94.5 ± 8.3%, n = 8) (Fig. 2C). The results in Figs 1 and 2 together demonstrate that activation of A1R but not A2AR or A3R leads to the depression of mGluR1 signalling in Purkinje cells.

View larger version (22K): [in this window] [in a new window]   Figure 2.  A2AR and A3R are not important for mGluR1 signalling depression A and B, neither an A2AR agonist (CGS21680, bath-applied at 150 nM, 7 cells) nor an A3R agonist (HEMADO, bath-applied at 30 nM, 7 cells) depresses DHPG (50 µM, 10 s)-evoked inward currents. C, comparison of the effects of the labelled test agents by the mean peak amplitudes at 3 min after bath application onset. Control, the normal saline (8 cells, reproduced from Fig. 1). CGS and HEMADO, data collected from the same cells used in A and B, respectively. NS, P > 0.05 compared with the control (rank score test).

A1R is coupled selectively to pertussis toxin (PTX)-sensitive G_i/o protein over other G proteins (Klinger et al. 2002). Thus, G_i/o protein could possibly mediate mGluR1 depression in cultured cerebellar Purkinje cells. However, this apparently conflicts with results from a previous study (Hirono et al. 2001), where activation of G_i/o protein via GABA_BR augmented mGluR1 signalling in Purkinje cells in cerebellar slices.

We therefore checked whether G_i/o protein could act on mGluR1 signalling in a different manner between culture and slice preparations. We stimulated G_i/o protein activation by GABA_BR with baclofen, a GABA_BR-selective agonist and measured its effect on the DHPG-evoked inward current. We used a saturating level of DHPG (500 µM (Tabata et al. 2002)) in this experiment to rule out the G_i/o protein-independent effect of baclofen which increases mGluR1's sensitivity to an unsaturating dose of DHPG (Tabata et al. 2004). Bath application of baclofen (1 µM, n = 7) but not the normal saline (n = 7) significantly augmented the peak amplitude of the inward current (Fig. 3A–C). This result suggests that G_i/o protein acts on mGluR1 signalling in the same manner in the culture and slice preparations.

View larger version (19K): [in this window] [in a new window]   Figure 3.  Gi/o protein activation via GABABR augments mGluR1 signalling A and B, bath application of a GABABR-selective agonist (baclofen, 1 µM, 7 cells, B) but not the normal saline (control, 7 cells, A) induces the augmentation of DHPG (10 s)-evoked inward currents. Only in these experiments, DHPG was applied at a saturating dose (500 µM, see Results for further explanation). C, comparison of the effects of the normal saline and baclofen by the mean peak amplitudes at 12 min after bath application onset. Data were collected from the same cells used in A and B. **P < 0.01 compared with the control (rank score test).

View larger version (31K): [in this window] [in a new window]   Figure 4.  A1R-mediated mGluR1 signalling depression does not require Gi/o protein A, pretreatment with a Gi/o protein uncoupler (PTX, 500 ng ml–1, for 16 h or longer, 7 cells) does not abolish the R-PIA (50 nM, bath-applied)-induced depression of DHPG (50 µM, 10 s)-evoked inward currents. B, comparison of the effects of the labelled test agents by the mean peak amplitudes at 12 min after bath application onset. Control, the normal saline without the PTX pretreatment (8 cells, reproduced from Fig. 1). R-PIA, R-PIA (50 nM) without the PTX pretreatment (7 cells, reproduced from Fig. 1). PTX + R-PIA, data collected from the same cells used in A. **P < 0.01 and NS, P > 0.05, respectively (rank score test). C, R-PIA (500 nM, 9 cells) but not the normal saline (control, 11 cells) induces an inwardly rectifying current, which is suppressed by the PTX pretreatment (500 ng ml–1, for 16 h or longer, 9 cells, PTX + R-PIA). Each plot indicates the mean (black line) and S.E.M. (grey lines) of the I–V relations of a current induced by the labelled agent that was extracted as a difference between the total currents evoked by voltage ramps before and after a 2 min local application of the agents. The Purkinje cells were voltage clamped in a ruptured-patch whole-cell mode. Note that the R-PIA-induced current has a reversal potential (–58.3 ± 4.9 mV) close to the EK (–56.7 mV). D, comparison of the amplitudes at –130 mV of currents induced by the labelled test agents. Data were collected from the same cells used in C. NS, P > 0.05 and **P < 0.01, respectively (ANOVA and unpaired t test).

The R-PIA-induced GIRK current (see above) shows functional coupling between A1R and G_i/o protein in Purkinje cells. Thus, A1R could possibly mediate the augmentation of mGluR1-coupled responses like GABA_BR when A1R activates sufficient G_i/o protein to operate its subsequent signalling cascade that modulates mGluR1 signalling (e.g. phospholipase C (PLC)–IP₃R cascade; see Discussion). We found that A1R indeed exerts such an action when strongly stimulated. In this experiment, we used 2-chloroadenosine (2-CA), an A1R-preferring agonist which is much more water soluble than other synthetic A1R agonists and thus, can be given at a high dose to achieve massive A1R stimulation in neurons (Dittman & Regeher, 1996). A high dose (30 µM) of 2-CA augmented the inward current transiently at the early phase of drug application (n = 6, Fig. 5A). This augmentation was followed by depression that developed onward (Fig. 5A). The peak amplitude at 3 min after 2-CA onset (168.7 ± 30.8% of the basal level; Fig. 5C) was significantly larger than the control (94.5 ± 8.0%, n = 8). The peak amplitude at 12 min after 2-CA onset (55.8 ± 12.5%, Fig. 5C, right) was significantly smaller than the control (94.3 ± 9.3%, n = 8). Pretreatment with PTX (500 ng ml¹, over 16 h) abolished selectively the transient augmentation (n = 7, Fig. 5B). The peak amplitude was not augmented at 3 min after 2-CA onset (83.0 ± 6.3%; Fig. 5C, left) while it was significantly more reduced (69.0 ± 3.8%) than the control at 12 min after 2-CA onset (Fig. 5C, right). These results indicate that the G_i/o protein-independent depression of mGluR1 signalling masks the G_i/o protein-dependent augmentation unless A1R is stimulated strongly. The results in Figs 3–5 together clearly demonstrate that a G_i/o protein-independent pathway underlies the A1R agonist-induced depression of mGluR1 signalling.

View larger version (22K): [in this window] [in a new window]   Figure 5.  A1R exerts the opposite actions on mGluR1 signalling through Gi/o protein-dependent and -independent pathways A and B, bath application of a high dose of a potent AR agonist (2-CA, 30 µM) augments DHPG (50 µM, 10 s)-evoked inward currents transiently prior to inducing depression in PTX-untreated Purkinje cells (6 cells, A) but not in PTX-pretreated cells (500 ng ml–1, over 16 h, 6 cells, B). Test 3 min and Test 12 min, responses at 3 min and 12 min after 2-CA onset. C, comparison of the effects of the labelled test agents by the mean peak amplitudes at 3 min and 12 min after bath application onset. Control, the normal saline without the PTX pretreatment (8 cells, reproduced from Fig. 1). 2-CA, 2-CA without the PTX pretreatment, data collected from the cells used in A. PTX + 2-CA, 2-CA with the PTX pretreatment, data collected from the same cells used in B. *P < 0.05 and **P < 0.01 compared with the control, respectively (rank score test).

G_i/o protein inhibits cAMP production by adenylyl cyclase and is thereby negatively coupled to cAMP and its subsequent signalling cascade. We examined the sensitivity of mGluR1 signalling to a change in cAMP level, using forskolin (20 µM), an adenylyl cyclase activator. The peak amplitudes of DHPG-evoked inward currents at 6 min (107.7 ± 9.0% of the basal level, n = 7) and 12 min (99.5 ± 7.9%, n = 7) of forskolin onset were not significantly different from the control (92.0 ± 4.4%, n = 7 and 94.3 ± 9.3%, respectively) (Fig. 6A and B). The cAMP level should indeed be increased in this experiment because the same dose of forskolin augmented the hyperpolarization-activated, mixed cation current (I_h) that is sensitive to cAMP in cerebellar neurons (Saitow & Konishi, 2000) (Fig. 6C and D). These results suggest that cAMP-dependent mechanisms cannot explain mGluR1 signalling depression.

View larger version (33K): [in this window] [in a new window]   Figure 6.  cAMP insensitivity of mGluR1 signalling A, bath application of an adenylyl cyclase activator (forskolin, 20 µM) has little effect on the amplitude of DHPG (50 µM, 10 s)-evoked inward currents (7 cells). B, comparison of the effects of the normal saline (7–8 cells, reproduced from Fig. 1) and forskolin (7 cells, data collected from the cells used in A) by the mean peak amplitudes at 6 min and 12 min after bath application onset. NS, P > 0.05 (rank score test). C, forskolin (20 µM) indeed changes cAMP level as it augments Ih. Each set of superimposed traces indicates sample responses of a cell obtained before (grey thick line) and after (thin black line) a 2 min local application of forskolin. Schematics below the traces, voltage protocol. The Purkinje cells were voltage clamped in the ruptured-patch whole-cell mode. D, comparison of the steady-state amplitudes of the time-dependent components of Ih after application of the normal saline (Control, 11 cells) or forskolin (8 cells). **P < 0.01 compared with the control, rank score test.

A1R agonist-induced depression is specific for metabotropic glutamate signalling

To exclude a possibility that A1R-mediated mGluR1 signalling depression is an artefact, we checked the direct effect of R–PIA on Purkinje cells. For measuring the mGluR1-coupled inward current (Figs 1–7 and 10, except Fig. 4C and D), we used the Cs⁺-containing pipette solution. With this solution, local application of R-PIA (50 nM) little changed the holding current level and input resistance measured by voltage steps (Fig. 8A). The relative holding current levels to the basal value (–89.1 ± 18.1 pA) at 3–4 min and 13–14 min of R-PIA onset were 104.0 ± 4.3% and 96.4 ± 7.2%, respectively (n = 5, Fig. 8A and B). The relative input resistances to the basal value (282 ± 82 M) at 3–4 min and 12–13 min R-PIA onset were 94.7 ± 5.7% and 101.6 ± 5.1%, respectively (Fig. 8A and C). As well, local application of CCPA (500 nM, n = 3), 2-CA (30 µM, n = 3), or adenosine (400 nM, n = 4) did not significantly change the holding current level (basal value, –106.8 ± 13.6 pA; at 3–4 min of drug onset, 96.4 ± 5.7%, 101.6 ± 3.2%, and 100.2 ± 6.6%; at 12–13 min of drug onset, 96.0 ± 10.5%, 98.3 ± 4.3%, and 103.6 ± 11.5%, respectively) and input resistance measured by +5 mV voltage steps (basal, 236.5 ± 24.7 M; at 3–4 min of drug onset, 121.4 ± 17.1%, 92.9 ± 3.1%, and 104.2 ± 4.8%; at 12–13 min of drug onset, 128.7 ± 18.0%, 102.3 ± 4.7%, 108.4 ± 8.4%, respectively) (P > 0.05, unpaired t test for the raw data; Fig. 8D). Thus, the depression of the inward current is not attributable to a change in the background conductance, which could shift the actual holding potential imposed to the cell membrane and thereby could affect the amplitude of the inward current.

Bath application of R-PIA (50 nM, 3–13 min) did not change the peak amplitude of an ionotropic glutamate receptor-mediated inward current evoked by local application of AMPA (10 µM, 100 ms) (546.3 ± 150.6 pA, changed by 0.5 ± 5.7% at 12–13 min of R-PIA onset, n = 5) (Fig. 9A and B). This result again suggests that A1R activation does not shift the holding potential. It also suggests that A1R agonist-induced depression occurs specifically for metabotropic glutamate signalling but not for ionotropic glutamate signalling.

Physiological levels of adenosine depress mGluR1 signalling

We examined how physiological levels of the endogenous AR agonist adenosine affect mGluR1 signalling. In some brain regions, microdialysis detects 40 nM of adenosine when no drug is administered and 400 nM of adenosine following pharmacological inhibition of the adenosine uptake mechanisms (Ballarin et al. 1991). These values may indicate the range of extracellular concentrations of adenosine under physiological conditions. Adenosine at both levels (40 nM, n = 8, Fig. 10A and 400 nM, n = 7, Fig. 10B) depressed the DHPG-evoked inward current. The depression developed upon adenosine onset and lasted throughout a 12 min application. When compared at 12 min after application onset, the peak amplitude of the inward current (70.9 ± 5.6% of the basal level with 40 nM adenosine, 67.0 ± 5.7% with 400 nM adenosine) was significantly more reduced than the control (94.3 ± 9.3%, n = 8) (Fig. 10C). The extent of the depression did not display obvious adenosine dose dependency (see Discussion). The peak amplitude recovered almost completely to the basal level after the offset of 40 nM adenosine (Fig. 10A). These results suggest that A1R may mediate a depressant effect on mGluR1 signalling in response to adenosine in the cerebrospinal fluid under physiological conditions.

A1R activation also depresses mGluR1-coupled intracellular Ca²⁺ mobilization

Finally, we performed fluorometry to examine whether A1R activation depresses mGluR1-coupled intracellular Ca²⁺ mobilization that is mediated by downstream signalling molecules and effectors different from those of the mGluR1-coupled inward current (see Introduction).

DHPG evoked a rise in the [Ca²⁺]_i (Fig. 11). Since this response is due mostly to Ca²⁺ release from the intracellular store (Sato et al. 2004), frequent DHPG application in a Ca²⁺-free saline leads to store depletion. To minimize the influence of such rundown, we applied DHPG with a prolonged interval (13 min) in a Ca²⁺-containing saline (Figs 11–13). When co-applied with DHPG (50 µM), R-PIA depressed the peak amplitude of the [Ca²⁺]_i rise to 47.0 ± 30.8% of the basal level (n = 3, Fig. 11A). This depression became evident within 1–4.4 s after co-application onset (n = 3, Fig. 11A, ‘Superimposed’, note that the response to DHPG alone continues to grow for 10 s, whereas the response to co-application of R-PIA and DHPG decelerates after 4.4 s of drug onset). This result suggests that the A1R agonist-induced depression of mGluR1 signalling can develop within a few seconds.

View larger version (15K): [in this window] [in a new window]   Figure 11.  A1R-mediated depression is also seen for mGluR1-coupled intracellular Ca2+ mobilization A, co-applied R-PIA (50 nM, & R-PIA) reduces the DHPG (50 µM)-evoked [Ca2+]i rise in wild-type (WT) Purkinje cells. From left to right: sample responses of a cell to local application of the labelled agents (10 s from arrowheads) obtained consecutively at an interval of 13 min. Superimposed, the left two traces are superimposed and justified at application onset; note that the response to co-applied DHPG and R-PIA decelerates after 4.4 s of drug onset. The saline contained 2 mM Ca2+. Vertical calibration bars, change in F340/F380. B and C, R-PIA dose dependence of depression of the DHPG (5 µM, 10 s)-evoked [Ca2+]i rise. In this and the following figures, the saline contained 2 mM Ca2+. B, each set of traces indicates sample responses of a cell before (Basal) and after (Test) a 12 min local application of the labelled dose of R-PIA. C, mean peak amplitude of the [Ca2+]i rise after R-PIA application plotted against logarithmic-scaled R-PIA dose. n = 5–10 cells for each point.

View larger version (22K): [in this window] [in a new window]   Figure 13.  Genetic depletion of A1R abolishes A1R-mediated mGluR1 depression A–F, R-PIA or adenosine depresses the DHPG-evoked [Ca2+]i rise in WT Purkinje cells but not in A1R-KO cells without affecting the resting [Ca2+]i. A and D, each set of traces indicates sample DHPG (5 µM, 10 s)-evoked [Ca2+]i rises obtained from a cell before and after a 12 min local application of 50 nM R-PIA or 400 nM adenosine. B, C, E, and F, comparisons of the mean peak amplitudes of the [Ca2+]i rises and the mean resting [Ca2+]i after application of R-PIA (B and C) or adenosine (E and F). n = 7 WT cells (data reproduced from the previous figure) and n = 10 A1R-KO cells in B and C, and 17 WT cells and 10 A1R-KO cells in E and F. *P < 0.05 and NS, P > 0.05 compared with the WT cells (rank score test).

The R-PIA (50 nM, 12 min)-induced depression of the DHPG (5 µM, 10 s)-evoked [Ca²⁺]_i rise was resistant to treatment with a G_i/o protein inhibitor, PTX (500 ng ml^–1, over 16 h before the recording) or NF023 (10 µM; the cell was perfused with NF023 for >15 min prior to and throughout the recording) (Fig. 12A). The peak amplitudes after R-PIA application (50.6 ± 7.0% of the basal, n = 8 with PTX; 75.0 ± 5.5%, n = 9 with NF023) were significantly smaller than that of the control (108.7 ± 9.1%, n = 11) and not different from that of the untreated cells (64.5 ± 10%, n = 7) (Fig. 12B). This result supports the G_i/o protein-independence of A1R agonist-induced depression of mGluR1 signalling.

View larger version (17K): [in this window] [in a new window]   Figure 12.  A1R-mediated depression of mGluR1-coupled intracellular Ca2+ mobilization is independent of Gi/o protein or PKA A and B, R-PIA-induced depression of the DHPG (5 µM, 10 s)-evoked [Ca2+]i rise persists after inhibition of Gi/o protein. A, each set of traces indicates sample responses of a cell obtained before and after a 12 min local application of the normal saline (Control) or 50 nM R-PIA. PTX +, cell pretreated with PTX (500 ng ml–1) for >16 h before the recording. NF023 +, cell treated with NF023 (10 µM) continuously for >15 min prior to and throughout the recording. B, comparison of the mean peak amplitudes of the [Ca2+]i rises after application of the normal saline or R-PIA. N = 7–11 cells for each group. *P < 0.05, **P < 0.01, and NS, P > 0.05 (rank score test). C and D, inhibition of PKA does not mimic A1R-mediated depression. C, each set of traces indicates sample DHPG (5 µM, 10 s)-evoked [Ca2+]i rises of a cell obtained before and after a 12 min local application of DMSO (0.01% v/v, vehicle for KT5720, Control) or 1 µM KT5720. D, comparisons of the mean peak amplitudes of the [Ca2+]i rises after application of the test drugs. n = 8–9 cells for each group. NS, P > 0.05 compared with the control (rank score test).

We compared the effect of R-PIA on the DHPG (5 µM, 10 s)-evoked [Ca²⁺]_i rise between cells derived from wild-type (WT) mice and the A1R-KO mice. R-PIA depressed the peak amplitude in WT cells (64.5 ± 10.0%, n = 7) but not in A1R-KO cells (99.4 ± 8.7%, n = 7) (Figs 13A and B). This result provides genetic evidence for the involvement of A1R in the A1R agonist-induced depression of GluR1 signalling.

A previous study (Hirono et al. 2001) has suggested that in cerebellar Purkinje cells, G_i/o protein activation results in Ca²⁺ release from the intracellular stores (see Discussion), and this may underlie the modulation of mGluR1-coupled responses. However, such modulation cannot explain the R-PIA-induced depression of mGluR1 signalling studied here. The resting [Ca²⁺]_i did not much change after R-PIA treatment (50 nM, 12 min) in the WT cells (91.8 ± 2.8% of the basal level, n = 7), and this extent of change was not different from that of the A1R-KO cells (96.6 ± 2.2%, n = 10) (Fig. 13C). This result clearly shows that with a dose of R-PIA that depresses the mGluR1-coupled inward current and [Ca²⁺]_i rise, A1R does not mediate a change in the resting [Ca²⁺]_i.

Furthermore, a physiological dose (400 nM, 12 min) of adenosine depressed the peak amplitude of the DHPG (5 µM)-evoked [Ca²⁺]_i rise in WT cells (81.2 ± 5.6% of the basal level, n = 17) but not in A1R-KO cells (107.5 ± 11.6%, n = 10) (Fig. 13D and E) without affecting the resting [Ca²⁺]_i (102.0 ± 1.6% for the WT cells, 101.3 ± 1.6% for the A1R-KO cells, Fig. 13F). This result suggests the A1R-mediated depression of mGluR1-coupled Ca²⁺ mobilization may occur under physiological conditions.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
We found that several A1R agonists (R-PIA, Fig. 1B and D; CCPA, Fig. 1C and D; and 2-CA, Fig. 4A and B) depressed the mGluR1-coupled inward cation current in cerebellar Purkinje cells. The A2AR- and A3R-selective agonists (CGS21680 and HEMADO, respectively) failed to depress the inward currents (Fig. 2), suggesting that neither A2AR nor A3R mediates the depression. The involvement of A2BR could not be directly assessed for lack of a commercially available A2BR-selective agonist. However, A2BR does not seem to be important for the depression studied here because R-PIA and CCPA at the doses used (50 and 500 nM, respectively; Fig. 1B and C) should only minimally activate A2BR (affinities for these agonists are 4–40 µM (Poulsen & Quinn, 1998; Klotz, 2000; Fredholm et al. 2001)). Genetic depletion of A1R completely abolished the depression of the mGluR1-coupled [Ca²⁺]_i rise (Fig. 13). These results together unequivocally demonstrate that activation of A1R but not the other AR subtypes leads to the depression of mGluR1 signalling in Purkinje cells (we hereafter term this effect A1R–mGluR depression).

A1R has a high coupling selectivity to the G_i/o class of G proteins (Klinger et al. 2002). R-PIA evoked the G_i/o protein-coupled inwardly rectifying K⁺ (GIRK) currents (Tabata et al. 2005) (Fig. 4C and D), showing that A1R is indeed coupled to G_i/o proteins in Purkinje cells. Nevertheless, activation of GABA_BR, another G_i/o protein-coupled receptor failed to mimic A1R-mGluR1 depression (Fig. 3). A1R–mGluR1 depression was resistant to G_i/o protein inhibitors (Figs 4A and B, 5B and C and 12A and B). These results strongly suggest that A1R–mGluR1 depression does not require G_i/o proteins.

We further assessed the involvement of signalling cascades downstream of the major G proteins. Prolonged application of forskolin had little effect on the amplitude of the inward current (Fig. 6A and B) although it indeed facilitated cAMP production by adenylyl cyclase (Fig. 6C and D). A PKA inhibitor had little effect on the amplitude of the mGluR1-mediated [Ca²⁺]_i rise (Fig. 12C and D). Thus, the cAMP-PKA signalling cascade is not responsible for mGluR1 signalling depression. These results also suggest that A1R does not exert the depression via the subunit of G_i/o and G_s proteins that regulate adenylyl cyclase. In certain cell types, the subunit complex of G_i/o protein may weakly activate PLC (for review, see (Selbie & Hill, 1998)) that is coupled to Ca²⁺ release from the IP₃R-equipped intracellular stores. A previous study (Hirono et al. 2001) has suggested that in cerebellar Purkinje cells, G_i/o protein activation results in Ca²⁺ release from the intracellular stores, and this may underlie the modulation of mGluR1-coupled responses. However, such G_i/o protein/Ca²⁺-dependent modulation cannot explain A1R-mGluR1 depression, because with the dose of R-PIA used, there was no change in the resting [Ca²⁺]_i (Fig. 13C). Moreover, R-PIA did not evoke the G_q protein-mediated inward current (Hartmann et al. 2004) (Fig. 7). Thus, A1R should not affect directly the mGluR1–G_q protein cascade that mediates the inward current and [Ca²⁺]_i rise. These results together suggest that A1R mediates mGluR1 signalling depression independently of the major G proteins and their subsequent signalling cascades. To our knowledge, all classical A1R-coupled neuronal responses described so far are mediated by G_i/o protein (Fredholm et al. 2001). A1R–mGluR1 depression may represent the first example of G_i/o protein-independent neuronal function of native ARs in central neurons.

Under the condition used for measuring the A1R-mediated depression of the inward current (with Cs⁺-containing pipette solution), the A1R agonists did not affect the background conductance (Fig. 8) and ionotropic glutamate receptor current (Fig. 9). These results suggest that A1R–mGluR1 depression results from specific modulation of mGluR1 signalling.

A1R activation depressed both the inward current (Fig. 1) and the [Ca²⁺]_i rise (Fig. 11) which are mediated by different downstream signalling molecules and effectors (see Introduction) without the aid of the major G proteins, the primary diffusible messengers linking GPCRs to remote targets (see above). Thus, A1R–mGluR1 depression might depend on a local molecular interaction that modulates the early step of the mGluR1 signalling cascade. The detailed molecular dissection of mechanisms linking A1R and mGluR1 is outside the scope of this study. Here we discuss some possibilities implied by the existing observations. There are increasing reports (Jordan & Devi, 1999; Bouvier, 2001; Ciruela et al. 2001; Ferre et al. 2002; Tabata et al. 2004; Kubo & Tateyama, 2005) that various GPCRs form hetero-oligomers in which one constituent modulates the other, presumably through direct interaction. Some studies (Ciruela et al. 2001; Ferre et al. 2002) suggests that A1R can form complexes with and functionally interact with mGluR1 in the heterologous system. Such direct interaction could occur in Purkinje cells, whereas it is uncertain at present. In an attempt to assess the possibility of complex formation between A1R and mGluR1 in mouse cerebellar neurons, we performed immunoprecipitation assay in the synaptosomal fraction of the cerebellum lysate (Supplemental text and Supplemental Fig. 1). This attempt was hindered because the total amount of A1R protein in the starting material was already low (Supplemental Fig. 1A) and decreased below the threshold of detection after fractionation (Supplemental Fig. 1B). The possibility of complex formation should be re-addressed in future with new tools and techniques. Another possible mechanism is that non-G protein intermediate molecules link A1R and mGluR1. Recently, various transmembrane and cytosolic proteins have been found to interact with and modulate GPCRs (Bockaert et al. 2004). The involvement of such a protein remains to be explored in future.

A1R could mediate the G_i/o protein-independent depression as well as G_i/o protein-dependent augmentation of mGluR1 signalling (Fig. 5). Thus, the net effect of an A1R agonist should be determined as a balance between these opposing actions. The augmentation would obscure the depression when A1R is strongly activated (Fig. 11C). This may explain why CCPA had a weaker depressant effect than R-PIA (Fig. 1B–D). CCPA might induce a higher extent of augmentation than R-PIA, because CCPA is thought to be slightly more potent than R-PIA at the concentrations used (affinities for A1R, 0.4–0.8 nM and 1.17–2 nM, respectively (Poulsen & Quinn, 1998; Klotz, 2000; Fredholm et al. 2001)).

The endogenous AR agonist adenosine depressed mGluR1 signalling at both the lowest and highest levels measured in the extracellular fluid in some brain regions (40–400 nM (Ballarin et al. 1991); Fig. 10A). This result suggests that the extracellular fluid always contains a high enough level of adenosine to depress mGluR1 signalling. The depression did not display desensitization throughout a prolonged application of adenosine (Fig. 10). Therefore, adenosine may tonically depress mGluR1 signalling to a certain degree under physiological conditions in situ. The extent of depression was not much different between 40 nM and 400 nM adenosine (Fig. 10C). One possibility is that A1R is almost fully activated by adenosine at both doses because A1R's dissociation constant for adenosine is 10 nM (Abbracchio et al. 1997). Another possibility is that although a larger fraction of the A1R population is activated by adenosine at 400 nM than at 40 nM, depression is compromised at the higher dose as seen with higher doses of R-PIA (Fig. 11C, see above).

When a high dose (400 nM) of adenosine was applied, the amplitude of mGluR1-mediated response recovered only partially after adenosine offset (Fig. 10B). This result indicates that massive A1R activation might result in a long-term change of mGluR1 signalling under physiological conditions. In addition, the recovery was faster and more complete after the offset of 40 nM adenosine (Fig. 10A) than after the offset of a comparable dose (50 nM) of R-PIA (Fig. 1B). This could be due partly to ecto-/plasma adenosine deaminases that degrade adenosine but not the synthetic agonist (Franco et al. 1997; Deussen, 2000).

It was impossible to distinguish A1R–mGluR1 depression from the previously described G protein-dependent inhibition of transmitter release by presynaptic A1R (Dittman & Regeher, 1996) in cerebellar slices (our unpublished data). Our attempt to examine whether A1R antagonists (1,3-dipropyl-8-phenylxanthine (DPPX) and 8-cyclopentyl-1,3-dipropylxanthine) could abolish the depressant effect of endogenous adenosine on glutamate-evoked, mGluR1-mediated currents in the cerebellar slice was also hindered. These antagonist at their effective doses (1–10 µM for DPPX) turned out to have a side-effect to directly inhibit mGluR1-mediated responses in cultured Purkinje cells (our unpublished data). DPPX at a very low dose (100 nM) had little effect on glutamate-evoked currents in cerebellar slices, whereas its effect at higher doses could not be tested because of the side-effect (our unpublished data). Thus, the in situ assessment of the physiological relevance of A1R–mGluR1 depression is difficult at present and awaits the development of new experimental techniques. However, our results together with the previous studies at least suggest a possibility that A1R–mGluR1 depression may influence long-term depression (LTD) of parallel fibre–Purkinje cell (PF–PC) synapses, a form of synaptic plasticity crucial for cerebellar motor learning (Ito, 2002). LTD is induced following repetitive, synchronized transmission at PF–PC and climbing fibre–Purkinje cell (CF–PC) synapses. CFs are reported to release adenosine and/or its precursors in an activity-dependent manner (Do et al. 1991). A biochemical study (Balaban et al. 1984) shows that activity of 5'-nucleotidase which catalyses the precursors into adenosine increases around the PF–PC synapses following stimulation of CFs. Adenosine depressed the mGluR1-coupled [Ca²⁺]_i rise (Fig. 13D and E). mGluR1-coupled Ca²⁺ mobilization is a key event for inducing LTD (Ito, 2002), Therefore, CF-derived adenosine may regulate the inducibility of LTD through A1R–mGluR1 depression. Moreover, A1R–mGluR1 depression may also influence information flow carried by slow EPSPs at PF–Purkinje cell synapses, because adenosine reduces an inward current mediated by the channels responsible for slow EPSPs (Fig. 10).

Adenosine is released from various neurons and glia, and thus occurs ubiquitously throughout the brain (see Introduction). Co-localization of A1R and mGluR1 is seen in cortical neurons as well as cerebellar neurons (Ciruela et al. 2001). Therefore, A1R–mGluR1 depression may take place in various brain regions. The findings in this study together demonstrate a novel neuromodulatory action of adenosine in central neurons.

	Footnotes

 
K. Hashimoto and H. Kassai contributed equally to this work. This paper has online supplemental material.

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