Purinergic modulation of the excitatory synaptic input onto rat striatal neurons

Neuropharmacology 62 (2012) 1756e1766

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Purinergic modulation of the excitatory synaptic input onto rat striatal neurons Michael Tautenhahn a, Anna Leichsenring a, Ilenio Servettini a,1, Michael Pesic a, Beata Sperlagh b, Wolfgang Nörenberg a, *, Peter Illes a a b

Rudolf-Boehm-Institute of Pharmacology and Toxicology, University of Leipzig, D-04107 Leipzig, Germany Laboratory of Molecular Pharmacology, Institute of Experimental Medicine, Hungarian Academy of Sciences, H-1083 Budapest, Hungary

a r t i c l e i n f o

a b s t r a c t

Article history: Received 23 September 2011 Received in revised form 21 November 2011 Accepted 1 December 2011

There is no in situ evidence hitherto for a modulation by ATP of the glutamatergic excitatory transmission onto medium spiny neurons (MSNs) in the rat striatum. In order to resolve this question, we used the patch-clamp technique in brain slice preparations to record excitatory postsynaptic currents (EPSCs) evoked by intrastriatal electrical stimulation and applied N-methyl-D-aspartate (NMDA) or a-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) to activate transmembrane currents of MSNs. In the absence of external Mg2þ, ATP caused a higher maximum inhibition of the EPSCs than adenosine. Only P1 (A1), but not P2 receptor antagonists interfered with the effects of both ATP and adenosine. Moreover, A1 receptor antagonists were less potent in blocking the inhibition by ATP than that by adenosine. Eventually, adenosine deaminase (ADA) almost abolished the adenosine-induced inhibition, but only moderately decreased the ATP-induced inhibition. Antagonists of A1 receptors (but not of P2 receptors) counteracted the depression by ATP of the current responses to exogenous NMDA, without altering those to AMPA. It is suggested that ATP indirectly, via its degradation product adenosine, stimulates presynaptic inhibitory A1 receptors situated at glutamatergic nerve terminals of striatal afferents; these nerve terminals are devoid of P2 receptors. However, ATP, in contrast to adenosine, also activates postsynaptic A1 receptors at the MSN neurons themselves. The resulting negative interaction with NMDA receptors requires localized extracellular catabolism of ATP by ectonucleotidases. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Striatal medium spiny neurons NMDA receptors ATP Adenosine Presynaptic purinergic modulation Postsynaptic purinergic modulation

1. Introduction Adenosine may either leave the intracellular space by a nucleoside transporter (Deussen, 2000) or may be generated by the enzymatic breakdown of extracellular ATP (Zimmermann, 2006). ATP itself is released from neurons and glial cells by exocytosis or

Abbreviations: ADA, adenosine deaminase; AMPA, (S)-a-amino-3-hydroxy-5methyl-4-isoxazolepropionic acid; AP-5, D,L-2-amino-5-phosphonopentanoic acid; CCPA, 2-chloro-N6-cyclopentyladenosine; CSC, 8-(3-chlorostyryl)caffeine; DPCPX, 8-cyclopentyl-1,3-dipropylxanthine; EPSC, excitatory postsynaptic current; GABA, g-aminobutyric acid; IPSC, inhibitory postsynaptic current; MRS 1220, N-[9-chloro2-(2-furanyl)[1,2,4]-triazolo[1,5-c]quinazolin-5-yl]benzene acetamide; MRS 2768, uridine-50 -tetraphosphate; MSN, striatal medium spiny neuron; NBQX, tetrahydro6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide; NMDA, N-methyl-D-aspartate; PD, Parkinson’s disease; PPADS, pyridoxalphosphate-6-azophenyl-20 ,40 -disulfonic acid; RB-2, reactive blue-2; 8-SPT, 8-(p-sulphophenyl)-theophylline. * Corresponding author. Rudolf-Boehm-Institute of Pharmacology and Toxicology, Haertelstrasse 16-18, D-04107 Leipzig, Germany. Tel.: þ49 341 9724605; fax: þ49 341 9724609. E-mail address: [email protected] (W. Nörenberg). 1 Present address: Department of Biotechnologies and Biosciences, University of Milano-Bicocca, I-20126 Milano, Italy. 0028-3908/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2011.12.001

nucleotide transporters but also by passing the damaged plasma membrane of injured cells (Illes and Alexandre, 2004; Burnstock, 2007). Four G protein-coupled adenosine receptors have been cloned and functionally identified to date, and are referred to as P1 receptors (Ralevic and Burnstock, 1998) consisting of the A1, A2A, A2B and A3 subtypes (Fredholm et al., 1994, 2011). In contrast, ATP activates seven subunits of ligand-gated cationic channels (P2X1e7), forming homo- or heteromeric receptor trimers, as well as eight different P2Y receptor subtypes (P2Y1e2, P2Y4, P2Y6, P2Y11e14) (Khakh et al., 2001; Abbracchio et al., 2006). The predominant neuronal type within the striatum is the GABAergic medium spiny projection neuron (MSN), which accounts for about 95% of the striatal cell population (Tepper and Bolam, 2004). Two subtypes of these neurons have been described (Kawaguchi et al., 1995). One subtype projects to the substantia nigra and internal segment of the globus pallidus forming a direct pathway to the thalamus and cortex. It contains substance P/dynorphin in addition to GABA (Parent and Hazrati, 1993; Latini et al., 1996). The other subtype targets the external segment of the globus pallidus forming an indirect pathway to analogous brain regions as the direct one, and contains enkephalin/ GABA. The cellular localization of the A2A receptor mRNA closely

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matches that of the dopamine D2 receptor mRNA, being expressed in striatopallidal projection neurons, which also express enkephalin (Schiffmann et al., 1991; Ongini and Fredholm, 1996). A2A receptor antagonists were shown to improve the motor symptoms of Parkinson’s disease (PD), by correcting the misbalance between the direct and indirect output pathways; they were also suggested to protect dopaminergic neurons from degeneration causing PD (Schwarzschild et al., 2006; Jenner et al., 2009). Whereas A2A/D2 receptors are co-localized at one subclass of MSNs, A1/D1 receptors are supposed to occur together at the other subclass of this cell type (Rivkees et al., 1995; Burnstock et al., 2011). Further, cholinergic striatal interneurons express both A2A and D2 receptors jointly modulating MSN function via the release of acetylcholine (Tozzi et al., 2011). In addition to postsynaptic A1 and A2A receptors situated at the cell bodies of MSNs and cholinergic interneurons, presynaptic A1 and A2A receptors appear to alter the dopaminergic, glutamatergic, GABAergic and cholinergic input to striatal neurons (Fredholm and Dunwiddie,1988; Latini et al.,1996; Flagmeyer et al., 1997; Pang et al., 2002; Wirkner et al., 2004; Ciruela et al., 2006). Although there is a wealth of information on the effect of adenosine in the striatum, our knowledge on the effects of striatal ATP is rather limited. In the rat striatum, mRNAs and proteins for a number of P2X and P2Y receptor-types were detected (Norenberg and Illes, 2000; Amadio et al., 2007). Since ATP was depolarizationdependently released from cultured striatal neurons (Zhang et al., 1988), as well as synaptosomes prepared from cholinergic interneurones of the striatum (Richardson et al., 1987) and because ectoenzyme activity for the breakdown of extracellular ATP was present at least in synaptosomes (James and Richardson, 1993), several important prerequisites for a possible neurotransmitter role of ATP in the striatum seem to be met. Notwithstanding this evidence, functional P2X/P2Y receptors were not found to be present at the GABAergic MSNs and cholinergic interneurons of the rat striatum by electrophysiological methods (Scheibler et al., 2004). Therefore, as an extension of this early in situ study, we investigated whether ATP or its degradation product adenosine modulates the glutamatergic excitatory input to striatal neurons at the pre- or postsynaptic level, in spite of being inactive as a neurotransmitter by itself. 2. Methods 2.1. Brain slice preparation and patch-clamp recording Striatal slices of young Wistar rats (10e14 days old) were prepared as previously described (Norenberg et al., 1997), according to the guidelines, and with the approval, of the Animal Care Committee at the Medical Faculty of the University of Leipzig. Experiments were carried out at room temperature (22e24  C) in a recording chamber (300e400 ml volume) continuously superfused (3 ml/min) with artificial cerebrospinal fluid (ACSF). Neurons were visualised by means of an upright microscope (40 water immersion objective) combined with Nomarski-type differential interference contrast (DIC optics). The ACSF had the following composition (mM): NaCl 126, KCl 2.5, NaH2PO4 1.2, CaCl2 2.4, NaHCO3 26 and glucose 11; saturated with 95% O2 and 5% CO2 (pH 7.4). Membrane currents and membrane potentials were recorded by means of a patch-clamp amplifier (Axopatch 200B, Axon Instruments) using patch pipettes (4e8 MU resistance) filled with an intracellular solution of the following composition (mM); K-gluconate 140, NaCl 10, MgCl2 1, HEPES 10, EGTA 11, Mg-ATP 1.5 and Li-GTP 0.3; pH 7.3 adjusted with KOH. The liquid junction potential (VLJ) between the bath and pipette solutions at 22  C was calculated as in previous experiments (Scheibler et al., 2004); it was found to be 14 mV. Membrane potential values were corrected for this amount of liquid junction potential. The holding potential was set at the calculated chloride equilibrium potential (ECl ¼ 61 mV) by choosing a pipette potential (Vp) of 47 mV. Striatal MSNs included in the data were selected on the basis of having 12e18-mm diameters (15.7  0.1 mm) and a stable resting membrane potential of at least 75 mV (77.1  0.9 mV; n ¼ 229 each) (Calabresi et al., 1987). The seal resistance was between 2 and 7 GU. Compensation of capacitance (Cm) and series resistance (Rs) was achieved with the inbuilt circuitry of the amplifier. After gaining whole-cell access, Rs was 16.6  0.01 MU (n ¼ 397). When Rs changed by more than 15% from the beginning until the end of the experiments, the cell was omitted from evaluation.

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Currents were filtered at 1 kHz with the inbuilt lowpass-filter of the patchclamp amplifier. Data were then sampled at 3 kHz and stored on-line with a PC using the pClamp 10.0 software package (Molecular Devices) that was also used for data analysis. 2.2. Electrical stimulation and application of drugs Synaptic currents were evoked by rectangular electrical pulses (0.1 Hz, 0.1 ms) delivered by a Grass S88 stimulator (Grass Technologies) via bipolar tungsten electrodes inserted into the slice 200e400 mm away from the neuron recorded from. The stimulation strength was chosen so that excitatory postsynaptic currents (EPSCs) of half-maximal amplitude were obtained. Drugs supposed to interfere with synaptic currents were applied by using three-way taps to replace the superfusion medium with another one of a modified composition. At the flow rate of 3 ml/min about 30 s were required until the drugs reached the bath. The effects of tetrodotoxin (TTX), ionotropic glutamate receptor and GABAA receptor antagonists, as well as Mg2þ were determined by comparing the average peak amplitudes of 6 EPSCs measured immediately before starting the superfusion of these compounds, with the average peak amplitudes of 6 EPSCs measured 10 min after compound superfusion. The concentrationeresponse curves of ATP and adenosine were evaluated by comparing the amplitudes of 6 EPSCs measured before agonist superfusion with 6 EPSCs measured 4 min after agonist superfusion. The same protocol was used when the effect of ADA was measured together with ATP or adenosine. In interaction experiments, all purinoceptor and ionotropic glutamate receptor antagonists were superfused for 35 min, before purinoceptor agonists were applied for an additional 4 min in the presence of these antagonists; once again the amplitudes of 6 EPSCs before and another 6 EPSCs after agonist application were compared with each other. The effects of adenosine and ATP were determined in separate experiments on the EPSC amplitude also during continuous electrical stimulation for 18 min. Another exception from the standard protocol was, when the effects of antagonists by themselves were determined for 45 min, by adding antagonists 3 min after the initial train of EPSCs was recorded. Drug effects were normalized with respect to the first averaged series of responses in a drug-free or antagonist-containing medium. In order to correct for time-dependent changes, experiments were performed in drug-free solutions throughout and the obtained percentage changes were compared to those obtained in drug-containing medium. In some experiments, N-methyl-D-aspartate (NMDA; 100 mM) or (S)-a-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA; 100 mM) dissolved in ACSF were applied for 1e2 s directly onto the cell by means of a rapid superfusion system (80e90 mmHg pressure) (DAD12, Adams and List). The intervals between subsequent drug-ejections were kept long enough (200 s) to obtain reproducible current amplitudes. The agonists were reapplied 8e12 times in total (T1eT12). The first response in a series (T1) was always omitted. Purinoceptor agonists (ATP, ADP, AMP, adenosine, uridine-50 -tetraphosphate [MRS 2768]) were applied after recording three responses to NMDA or AMPA either in the absence (T1eT3) or presence of ifenprodil, pyridoxalphosphate-6-azophenyl-20 ,40 -disulfonic acid (PPADS) or 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; T4eT6). The current amplitudes were normalized with respect to the averaged amplitudes at T2/T3 or T5/T6 obtained before the application of purinoceptor agonists. All changes were expressed as percentage inhibition at T5/T6 or T8/T9 and were compared with the control (drug-free) decrease of NMDA current amplitudes (pooled values of the percentage changes from T2/T3 to T5/T6 and T5/T6 to T8/T9). The effect of 2-chloro-N6-cyclopentyladenosine (CCPA) on the NMDA-induced currents either alone or in the presence of N-[9-chloro-2(2-furanyl)[1,2,4]-triazolo[1,5-c]quinazolin-5-yl]benzene acetamide (MRS1220) was measured in a similar manner as described for ATP and its antagonists. 2.3. Materials All drugs were purchased from Sigma-Aldrich with the exception of 8-SPT, CCPA, DPCPX DL-AP-5, ifenprodil, MRS 1220, MRS 2768, NBQX, and TTX which were from Tocris. The pH value of the ATP-containing ACSF superfused onto the MSNs was checked routinely and, when necessary, readjusted with NaOH to 7.4. 2.4. Statistics Means  S.E.M. are given throughout. Differences in means were tested for significance by one-way ANOVA followed by the HolmeSidak test. A probability level of 0.05 or less was considered to be statistically significant. The version 11.0 of the package SigmaPlot (Jandel Scientific) was used for all statistical evaluations.

3. Results 3.1. Involvement of NMDA, AMPA and GABAA receptors in excitatory synaptic currents of striatal neurons in Mg2þ-free bath solution In a first series of experiments, we confirmed and extended our previous results obtained on EPSC/IPSC complexes of MSNs

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(Wirkner et al., 2004). The main difference to this earlier work was that we kept the holding potential at 61 mV instead of 80 mV, and thereby at the calculated equilibrium potential of Cl. Thus, chances for a contamination of the EPSCs by GABAA receptormediated currents were minimized; the GABAA receptor antagonist bicuculline (10 mM) in fact did not inhibit the EPSCs (Fig. 1B). Therefore, all further recordings were made in a bicuculline-free ACSF, although we are aware of the fact that a minor contribution of GABAergic IPSCs to glutamatergic EPSCs due to non-optimal space-clamp conditions in brain slices cannot be completely excluded. However, an alternative possibility would have been to record in the continuous presence of bicuculline, which increased the neuronal excitability and distorted the shape and amplitude of EPSCs, because of repetitive discharges. In a Mg2þ-free bath medium and at the holding potential of 61 mV, intrastriatal stimulation-evoked EPSCs, with the mean amplitude of 483.6  13.1 pA (n ¼ 229). The large EPSC amplitudes and their prolonged duration indicated that on the one hand the Mg2þ block of the NMDA receptor-channels was relieved and on the other hand the release of glutamate was enhanced by Mg2þ-deficiency (see Fig. 1; Wirkner et al., 2004). TTX (0.3 mM) abolished the EPSCs (98.6  1.4% inhibition; n ¼ 5), which were also markedly depressed in a Ca2þ-free external medium (77.6  5.3% inhibition; n ¼ 5) (Fig. 1B). Thus, they were due to the exocytotic, Ca2þ-dependent release of a neurotransmitter initiated by conducted action potentials carried by Naþ. The percentage changes indicated throughout the paper refer to inhibitory effects, without explicitly stating it. As already mentioned,

bicuculline (10 mM) failed to modify the EPSC amplitude. Mg2þ (1.3 mM), causing an open channel block of NMDA receptor-channels (43.4  5.9%; n ¼ 8), and D,L-2-amino-5-phosphonopentanoic acid (AP-5; 50 mM), a competitive NMDA receptor antagonist (46.9  4.1%; n ¼ 7), both approximately halved the EPSCs (Fig. 1Ab, B; for the peak currents see inset). 1,2,3,4-Tetrahydro-6-nitro-2,3-dioxo-benzo[f] quinoxaline-7-sulfonamide (NBQX; 10 mM), a competitive AMPA/ kainate receptor antagonist, blocked the other half of the EPSC (40.9  6.6%; n ¼ 7) (Fig. 1Ac, B). The abolition of the EPSCs by a combination of D,L-2-amino-5-phosphonopentanoic acid (AP-5) and NBQX confirmed that the EPSC in a Mg2þ-free medium consists of NMDA and AMPA/kainate receptor-mediated fractions (Fig. 1Ad, B) (see Jiang and North, 1991). 3.2. Effect of ATP and adenosine on excitatory synaptic currents of striatal neurons We chose stimulation conditions, which evoked stable EPSCs under drug-free conditions, when trains of 6 stimuli at 0.1 Hz were delivered 0, 5 and 12 min, after starting stimulation (Fig. 2C). In analogous experiments, adenosine (30 mM) and ATP (300 mM) were applied for 5 min, with a subsequent 6 min washout period (Fig. 2A, B, C). The use of short trains of stimuli resulted in more stable EPSC amplitudes than during continuous electrical stimulation at 0.1 Hz for 12 min (Fig. 2C, inset). Under these latter conditions, there was a progressive timedependent decline of the EPSCs even in the absence of any drug. This led to a seemingly incomplete washout of the agonist-effects and

Fig. 1. Characterization of EPSC components at striatal medium spiny neurons mediated by ionotropic glutamate receptors. Focal, intrastriatal stimulation was used by applying 0.1ms pulses at 0.1 Hz and a stimulation strength evoking a half-maximal EPSC amplitude. For further methodological details including the evaluation protocols for this and all subsequent Fig. consult the Methods section. (A) Original recordings of two EPSCs evoked with 10-min intervals (Control; a). The first EPSC was induced before and the second 10 min after superfusing AP-5 (50 mM; b), NBQX (10 mM; c) and the combination of these antagonists (d). The insets in each panel show the pairs of EPSCs at a faster recording speed (see different time-scale). (B) A similar application schedule was used to determine the effects of tetrodotoxin (TTX; 0.3 mM), a Ca2þ-free medium, bicuculline (10 mM), Mg2þ (1.3 mM), AP-5 (50 mM), NBQX (10 mM), and AP-5 (50 mM) plus NBQX (10 mM). Percentage inhibition of EPSCs; mean  S.E.M. of 5e8 cells. *P < 0.05; statistically significant difference from controls.

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forced us to apply trains of stimuli rather than continuous stimulation in all subsequent experiments. Both adenosine (30 mM) and ATP (300 mM) depressed the EPSCs in every neuron investigated and with a rather small scatter around the mean value (Fig. 2C). Thus, there was no indication for a separate innervation of the supposedly enkephalinergic and dynorphinergic MSNs by adenosine (or ATP)-sensitive and adenosine (or ATP)-resistant glutamatergic fibres. Then, adenosine (0.3e300 mM) and ATP (1e3000 mM) were superfused for 5 min at each concentration to construct concentrationeresponse curves (Fig. 2A, B, D, E). The respective IC50 (6.1  2.3 and 43.9  14.2 mM) as well as Imax values (64.5  5.9 and 82.9  5.8%; n ¼ 3e8 each) were larger for ATP than for adenosine; at the same time the respective Hill coefficients did not change (ATP, 0.760  0.149; adenosine, 0.914  0.248). Thus, adenosine had a higher potency, but caused a smaller maximum inhibition of EPSCs than ATP. This may be due to the fact that ATP activates both presynaptic P1 and P2 receptors, and/or that it interferes with postsynaptic ionotropic glutamate receptors. In contrast, adenosine may activate presynaptically its own P1 receptors only, and may leave postsynaptic glutamate receptors unaltered. In the following experiments, equipotent concentrations of adenosine (30 mM) and ATP (300 mM) were used in combination with rather high antagonist concentrations, known to block agonist-effects (see e.g. Ralevic and Burnstock, 1998). 3.3. Interaction of ATP and adenosine with antagonists for purinoceptors and with drugs interfering with the enzymatic degradation of these agonists All antagonists tested caused a progressively developing blockade of the EPSC amplitude, when given alone (Fig. 3C). The

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investigated antagonists were the following: 8-(p-sulphophenyl)theophylline (8-SPT; 100 mM), a non-specific and membrane impermeable adenosine receptor antagonist; DPCPX (0.3 mM), a selective A1 receptor antagonist; 8-(3-chlorostyryl)caffeine (CSC; 3 mM), a selective A2A receptor antagonist; suramin (100 mM) and PPADS (100 mM), two non-selective P2 receptor antagonists, and reactive blue-2 (RB-2; 100 mM) a subtype-non-selective, preferential P2Y receptor antagonist. Because we applied agonists 35 min after antagonist superfusion, all antagonists caused a peak inhibition within this period of time, which did not increase with a longer-lasting superfusion (n ¼ 6e10). Adenosine (30 mM) caused the expected inhibition of 60.8  8.5% (n ¼ 6) (Fig. 3Ab, B). 8-SPT (100 mM; n ¼ 7) and DPCPX (0.3 mM; n ¼ 5) abolished the effect of adenosine; RB-2 at 100 mM, but not at 30 mM (n ¼ 7 each) inhibited the adenosine effect (Fig. 3Ac, d, B). However, it is noteworthy that this concentration of RB-2 also greatly depressed the EPSCs by itself (Fig. 3C). CSC (3 mM), suramin (30, 100 mM) and PPADS (30, 100 mM) were inactive against adenosine (Fig. 3B). Fig. 4 shows the effects of the above antagonists against ATP (300 mM; 59.5  4.7%; n ¼ 7). Interestingly, both 8-SPT (100 mM; 33.5  5.1%; n ¼ 6) and DPCPX (0.3 mM; 18.2  3.2%; n ¼ 6) depressed the effect of ATP, but did not abolish it (Fig. 4Ac, B), in discrepancy to their interaction with adenosine, which led to a complete blockade (Fig. 3Ac, B). In addition, RB-2 (100 mM; n ¼ 9) failed to alter the effect of ATP, in spite of depressing that of adenosine. It is interesting to note that DPCPX (0.3 mM) (27.3  3.8%; n ¼ 8) alone caused the same amount of inhibition as in combination with suramin (30 mM) (23.4  3.5%; n ¼ 8) or RB-2 (30 mM) (24.0  5.8; n ¼ 4) (Fig. 4Bb). Thus, ATP appears to be

Fig. 2. Concentrationeresponse relationships for the inhibitory effects of adenosine and ATP on the EPSC amplitudes of striatal medium spiny neurons. Focal, intrastriatal stimulation was used by applying 0.1-ms pulses at 0.1 Hz and a stimulation strength evoking a half-maximal EPSC amplitude. Two different types of stimulation were used. Whereas continuous stimulation with a frequency of 0.1 Hz led to a gradual decrease of the EPSC amplitudes (inset to C), trains of stimuli (6 stimuli at 0.1 Hz) repeated at intervals of 5e7 min resulted in stable EPSC amplitudes (A, B, C). Original recordings showing the adenosine (Ado; 30 mM) and ATP (300 mM)-induced inhibition of EPSCs with a complete recovery after washout, as well as the mean  S.E.M. inhibition determined in 4e5 experiments. The pre-drug responses are designed as controls. (D) Original recordings of individual EPSCs out of trains caused by the above stimulation protocol, before, during and after the application of increasing concentrations of adenosine and ATP. (E). Concentrationeresponse curves of the adenosine- and ATP-induced percentage inhibition of EPSC amplitudes. Means  S.E.M. of 3e8 cells. *P < 0.05; statistically significant difference from controls.

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Fig. 3. Interaction between adenosine on the one hand, and nucleoside or nucleotide receptor antagonists on the other, on EPSC amplitudes of striatal medium spiny neurons. Focal, intrastriatal stimulation was used by applying 0.1-ms pulses at 0.1 Hz and a stimulation strength evoking a half-maximal EPSC amplitude. (A) Original recordings of 3 EPSCs induced before, during and after the application of adenosine (30 mM), either in the absence (b) or in the presence of DPCPX (0.3 mM; c) or reactive blue-2 (RB-2; 100 mM; d), respectively. All antagonists were applied 35 min before starting an experiment. Time-dependent change of the EPSCs evoked with a similar stimulation protocol as in the interaction experiments (4 min interval between the 3 trains; a). (B) The effect of adenosine (30 mM) on the EPSCs is expressed as percentage inhibition. The time-dependent change in EPSC amplitudes are designated as controls. 8-(p-sulphophenyl)-theophylline (8-SPT; 100 mM), 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 0.3 mM), and the higher concentration of RB-2 (100 mM) counteracted the effect of adenosine. 8-(3-chlorostyryl)caffeine (CSC; 3 mM), suramin (30, 100 mM), pyridoxalphosphate-6-azophenyl-20 ,40 -disulfonic acid (PPADS; 30, 100 mM) and RB-2 (30 mM) failed to alter the adenosine-induced depression of EPSCs. Mean  S.E.M. of 6e10 cells. *P < 0.05; statistically significant difference from controls. **P < 0.05; statistically significant difference from the effect of adenosine alone. (C) Time-dependent inhibition by nucleoside and nucleotide antagonists applied for 30 min and no major recovery after washout for an additional 10 min. Average percentage inhibition for 5e7 cells and each antagonist. The S.E.M. of the mean is not indicated for better visibility.

degraded to adenosine, which then acts at presynaptic A1 (but not A2A) receptors to depress the release of glutamate onto MSNs (Calabresi et al., 1997). By contrast, a direct modulatory effect of ATP via presynaptic P2 receptors was missing. In a further approach, we tested the effects of ATP, ADP, AMP, adenosine, inosine, UTP and UDP, all at 300 mM (Fig. 5A; n ¼ 5e8). The inhibition of EPSCs was the strongest with ATP and somewhat less pronounced with adenosine. ADP and AMP also comparably depressed the EPSC amplitudes, whereas inosine was inactive. UTP and UDP caused the least inhibition. According to the original definition, ATP is a general P2X/P2Y agonist, whereas ADP stimulates P2Y1,12,13 receptors only. AMP and adenosine are adenosine receptor agonists. Inosine is the inactive metabolite of adenosine, generated by the enzyme ADA. UTP and UDP are pyrimidine nucleotides stimulating P2Y2,4,6 receptors (von Kugelgen, 2006). In spite of demonstrating the inhibitory effects of UTP/UDP on the EPSC amplitudes, the mechanism of action of these agonists was not investigated further. In view of the fact that ADA degrades adenosine but not ATP to the inactive inosine, we tested the two purinoceptor agonists in the presence of ADA (Fig. 5B). ADA (10 U/ml; n ¼ 6) abolished the adenosine (30 mM)-induced inhibition of EPSCs (n ¼ 5), but only moderately decreased the action of ATP (69.3  3.9%; n ¼ 4). This

finding agrees with similar data reported for hippocampal CA1 synapses at room temperature (Mendoza-Fernandez et al., 2000). An increase in ambient temperature from 22 to 24  C (our experiments) to 30.5  C (Cunha et al., 1998) appeared to account for the differential potency of ADA in interfering with ATP and adenosine, because the enzymatic activity is expected to increase at higher temperature values. Nonetheless, we conclude that, at least at room temperature, adenosine generated from ATP in close neighbourhood of the glutamatergic nerve terminals is more resistant to degradation by ADA than bath applied adenosine. Hence, ATP may act via its degradation products AMP and adenosine on presynaptic A1 receptors to decrease the release of glutamate onto the MSNs; the involvement of presynaptic P2 receptors was excluded by these experiments. 3.4. Interaction of ATP with antagonists for ionotropic excitatory amino acid receptors and adenosine A1 receptors The inhibition by ATP (300 mM; 54.2  4.1%; n ¼ 6) of the EPSCs was not altered either by pretreatment with AP-5 (50 mM; 50.8  4.4; n ¼ 7) to block NMDA receptors or with NBQX (10 mM; 51.8  4.1%; n ¼ 9) to block AMPA receptors (Fig. 6Aa, Ab, Ca). Thus, ATP, under these conditions, did not interact either with the AMPA/

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Fig. 4. Interaction between ATP on the one hand, and nucleoside or nucleotide receptor antagonists on the other, on EPSC amplitudes of striatal medium spiny neurons. Focal, intrastriatal stimulation was used by applying 0.1-ms pulses at 0.1 Hz and a stimulation strength evoking a half-maximal EPSC amplitude. (A) Original recordings of 3 EPSCs induced before, during and after the application of ATP (300 mM) either in the absence (b) or in the presence of DPCPX (0.3 mM; c) or reactive blue-2 (RB-2; 100 mM; d), respectively. All antagonists were applied 35 min before starting an experiment. Time-dependent change of the EPSCs evoked with a similar stimulation protocol as in the interaction experiments (4 min interval between the 3 trains; a). (Ba) The effect of ATP (300 mM) on the EPSCs is expressed as percentage inhibition. The time-dependent change in EPSC amplitudes are designated as controls. 8-(p-sulphophenyl)-theophylline (8-SPT; 100 mM) and 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 0.3 mM) counteracted the effect of ATP. 8-(3-chlorostyryl) caffeine (CSC; 3 mM), suramin (30, 100 mM), pyridoxalphosphate-6-azophenyl-20 ,40 -disulfonic acid (PPADS; 30, 100 mM) and RB-2 (30 mM) failed to alter the adenosine-induced depression of EPSCs. Mean  S.E.M. of 6e9 cells. (Bb) The reduction of the inhibitory ATP (300 mM) effect by DPCPX (0.3 mM) is not altered by co-applying suramin (30 mM) or RB-2 (30 mM). Mean  S.E.M. of 4e8 cells. *P < 0.05; statistically significant difference from controls. **P < 0.05; statistically significant difference from the effect of ATP alone.

kainate or the NMDA receptor-mediated components of the EPSC. However, after the pharmacological blockade of A1 receptors by DPCPX to interfere with adenosine generated by the enzymatic breakdown of ATP, such an interaction became unmasked, since ATP in the presence of DPCPX caused a smaller inhibition than in its absence (30.7  2.8%; n ¼ 7) (compare Fig. 6Ca with b). Moreover, ATP in the combined presence of DPCPX and NBQX also depressed the EPSCs to a smaller extent than in the absence of these antagonists (25.2  4.7%; n ¼ 7) (Fig. 6Bb, Ca, Cb). Eventually, in the combined presence of AP-5 and DPCPX, ATP had no effect at all, tentatively suggesting a partly postsynaptic interaction between adenosine, derived from its mother compound ATP, and NMDA receptors of MSNs (Fig. 6Ba, Cb). This inhibitory interaction appears to be mediated by postsynaptic A1 receptors. 3.5. Modulation by ATP and adenosine of NMDA- and AMPAinduced inward currents; interaction with NMDA receptor antagonists, as well as P2 and P1 receptor antagonists Then, a purported postsynaptic interaction between ATP/adenosine on the one hand and NMDA/AMPA on the other was investigated and found to hold true for NMDA receptors only (compare representative experiments in Fig. 7Aa, b with Ba, b). The respective agonists for ionotropic glutamate receptors were applied at

a concentration of 100 mM in regular intervals (200 s) onto MSNs to induce inward current responses. ATP (300 mM), in contrast to ADP, AMP and adenosine (all 300 mM), blocked the NMDA currents by 26.7  3.5% (n ¼ 14; Fig. 7Aa, b, B). All cells reacted to ATP with a relatively constant amount of inhibition excluding the possibility that the two populations of MSNs (supposedly enkephalinergic and dynorphinergic) exhibit a differential sensitivity to this agonist. Thereafter, we investigated, whether the effect of ATP relies on the interaction with the NR2B subunit of NMDA receptors; this was, however, not the case, because ATP continued to act in the presence of the selective NR2B blocker ifenprodil (10 mM; Fig. 7Ac). Then, we studied the effects of the two non-selective P2X/Y receptor antagonists PPADS and suramin. ATP did not interact with PPADSsensitive P2 receptors (P2X1,2,3,4,7 and P2Y1,4,6,13; von Kugelgen, 2006; Jarvis and Khakh, 2009), given the inability of PPADS (100 mM) to antagonize the ATP-induced inhibition of NMDA responses (Fig. 7Ac). Similarly, suramin (100 mM), with selectivity for P2X1,2,3,5,7 and P2Y1,2,6,11,12,13 receptors (von Kugelgen, 2006; Jarvis and Khakh, 2009), also failed to inhibit the depression of NMDA currents by ATP. In addition, DPCPX (0.3 mM) both alone and in the presence of PPADS abolished the ATP effect, suggesting that in contrast to ATP itself, its degradation product adenosine may interact with NMDA receptors via A1 receptor stimulation (Fig. 7Ac). It is noteworthy that ifenprodil, PPADS and suramin, but not DPCPX

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of rapid adenosine uptake or a massive desensitization of the A1 receptor. We assumed that the effect of CCPA on local application of a rather high concentration of 1 mM is due to a non-specific effect mediated by other sites than the desensitized A1 receptors (for A3 receptors see Table 3 in Fredholm et al., 2011). In fact, the highly selective A3 receptor antagonist N-[9-chloro-2-(2-furanyl) [1,2,4]-triazolo[1,5-c]quinazolin-5-yl]benzene acetamide (MRS 1220) had no effect when given alone, but inhibited the facilitatory effect of CCPA (Fig. 7Ad). In agreement with the inability of suramin to antagonize the effect of ATP (Fig. 7Ac), the highly selective P2Y2 receptor agonist MRS 2768 (100 mM) did not interfere with ATP (4.7  10.9%; n ¼ 6). The A2A receptor agonist CGS 21680 depressed the NMDA currents in some but not all striatal neurons investigated (Fig. 7 Ad); CGS 21680 (0.1 mM) depressed NMDA currents by 41.9  4.1% in 6 MSNs, whereas in the residual 3 MSNs there was no inhibition at all. The effect of CGS 21680 was antagonized by the selective A2A receptor antagonist CSC (Norenberg et al., 1997). Eventually, ADP (300 mM) with selectivity for P2Y1,12,13 receptors failed to interact with NMDA (Fig. 7Ad), in accordance with the inability of PPADS to abolish the ATP effect. ATP, ADP, AMP and adenosine (all 300 mM) did not alter the current response to AMPA (100 mM) (Fig. 7Ba, b for ATP; n ¼ 5; otherwise not shown). Thus, the indirect interaction between ATP and NMDA via adenosine-stimulated A1 receptors may add a postsynaptic component to the inhibitory effect of ATP on EPSCs. In contrast, the AMPA/kainate fraction of the EPSCs appears not to be modulated by ATP or adenosine. 4. Discussion

Fig. 5. Inhibition by nucleotides and nucleosides of the EPSC amplitudes of striatal medium spiny neurons. Focal, intrastriatal stimulation was used by applying 0.1-ms pulses at 0.1 Hz and a stimulation strength evoking a half-maximal EPSC amplitude. (A) The effects of ATP, ADP, AMP, inosine, UTP and UDP (all at 300 mM), and of adenosine (30 mM) on the EPSCs are expressed as percentage inhibition. The timedependent changes in EPSC amplitudes are designated as controls. (B) Effects of adenosine (30 mM) and ATP (300 mM) in the absence and presence of adenosine deaminase (10 U/ml). Mean  S.E.M. of 5e9 cells. *P < 0.05; statistically significant difference from controls. **P < 0.05; statistically significant difference from the effect of ATP alone.

depressed the effect of NMDA, when applied alone, i.e. in the absence of ATP. In contradiction to the antagonistic properties of DPCPX on the ATP and adenosine effects, we found that adenosine and AMP (300 mM each) did not modulate the NMDA currents in MSNs (Fig. 7Ad; Wirkner et al., 2000). This result agrees with our earlier finding that adenosine was able to inhibit the NMDA currents in MSNs only after the blockade of nucleoside uptake (Norenberg et al., 1997). Locally applied 2-chloro-N6-cyclopentyladenosine (CCPA; 1 mM), a selective A1 receptor agonist even facilitated the NMDA responses (35.6  6.1%; n ¼ 6; Fig. 7Ad), strengthening the previous observation that CCPA did not modify the NMDA-induced current responses on bath application. In view of the facilitation of NMDA responses by CCPA and the failure of both adenosine and AMP to act on these currents, it may be speculated that exogenous A1 receptor agonists do not depress NMDA currents either because

The main finding of this paper is that exogenous ATP depresses striatal EPSCs by a combined pre- and postsynaptic effect, whereas exogenous adenosine causes inhibition via presynaptic receptors only. Adenosine A1 receptors are somewhat weakly expressed in striatal neurons (Rivkees et al., 1995), but are present on all types of afferent fibres to MSNs (Fredholm and Dunwiddie, 1988; Flagmeyer et al., 1997; see Introduction). The presence of presynaptic, inhibitory P2Y receptors in the striatum operating as targets of extracellular ATP is less clear. It has been shown that the electrically evoked release of dopamine in rat neostriatal brain slices was depressed by ATP in a manner reversed by the P2Y preferential antagonists reactive blue-2 and cibacron blue 3 GA (Trendelenburg and Bultmann, 2000). In contrast, microdialysis studies in awake rats demonstrated an increased extracellular dopamine level through stimulation of P2Y receptors in the rat striatum (Zhang et al., 1995; Krugel et al., 2001). However, under in vivo conditions, direct effects at dopaminergic nerve terminals may be obscured by indirect effects via the glutamatergic and/or GABAergic neurons integrated into functional networks. More convincingly, the release of glutamate from purified nerve terminals of the rat striatum was inhibited by P2Y1,2,4 receptors (Rodrigues et al., 2005). In addition to the biochemical evidence, electrophysiological findings also supported the notion that nucleotides per se can inhibit neurotransmission in the CNS and that, in the rat hippocampus, conversion to adenosine is not required (MendozaFernandez et al., 2000). It was hypothesized that there may be a presynaptic nucleotide receptor that inhibits neurotransmitter release, that is activated directly by ATP and ATP analogues, and is insensitive to most ATP receptor antagonists, although selective A1 receptor antagonists such as 8-cyclopentyltheophylline block the nucleotide effects (Shinozuka et al., 1988; Smith et al., 1997). In the present experiments, we recorded EPSCs of rat neostriatal slices in a Mg2þ-free superfusion medium, which relieves the Mg2þ block of NMDA receptor-channels and thereby favours the

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Fig. 6. Interaction between ATP and NMDA or AMPA/kainate receptor antagonists on EPSC amplitudes of striatal medium spiny neurons. Focal, intrastriatal stimulation was used by applying 0.1-ms pulses at 0.1 Hz and a stimulation strength evoking a half-maximal EPSC amplitude. (A) Original recordings of 3 EPSCs induced before, during and after the application of ATP (300 mM) in the presence of AP-5 (50 mM; a) or NBQX (10 mM; b). (B) Original recordings of 3 EPSCs induced before, during and after the application of ATP (300 mM) in the presence of DPCPX (0.3 mM) plus AP-5 (50 mM; a) or DPCPX (0.3 mM) plus NBQX (10 mM; b). All antagonists were applied 35 min before starting an experiment. (Ca) The effect of ATP (300 mM) on the EPSCs is expressed as percentage inhibition. The time-dependent changes in EPSC amplitudes are designated as controls. Both AP-5 (50 mM) and NBQX (10 mM) failed to alter the ATP-induced depression of EPSCs. Mean  S.E.M. of 7e9 cells. (Cb) DPCPX (0.3 mM) decreased the effect of ATP (300 mM) on the EPSCs; this action was not altered by the additional presence of NBQX (10 mM), but was abolished by the additional presence of AP-5 (50 mM). Mean  S.E.M. of 7 cells each. *P < 0.05; statistically significant difference from controls (in Cb from DPCPX alone). **P < 0.05; statistically significant difference from the effect of ATP alone.

NMDA-component of the EPSCs. Electrically evoked excitatory postsynaptic potentials (EPSPs) in rat MSNs were shown to be inhibited by adenosine in an 8-cyclopentyl-1,3-dimethylxanthineantagonizable manner, indicating the involvement of presynaptic A1 receptors situated at glutamatergic afferent fibres (Calabresi et al., 1997). These inhibitory A1 receptors could also be activated by the release of endogenous adenosine induced by superfusion of the brain slices with a glucose-free medium. Similarly, both adenosine and aglycemia depressed inhibitory postsynaptic currents (IPSCs) mediated by the release of GABA from intrastriatal afferents (Centonze et al., 2001). It is interesting to note that when striatal slices were taken from rats which underwent transient forebrain ischemia in vivo, an A1 receptor-mediated depression of the EPSC amplitudes was observed in large aspiny neurons (Pang et al., 2002). Under the Mg2þ-free conditions of our own experiments, both adenosine and ATP concentration-dependently inhibited the EPSC amplitudes. ATP had a higher IC50 value, and a larger Imax than adenosine. We asked ourselves, about the reason of the larger maximum inhibition of the EPSCs by ATP than by adenosine, and searched, therefore, for the purinoceptors involved. In perfect agreement with data collected on the hippocampus (see above), we found that P2 receptor antagonists did not interfere with the presynaptic inhibition by either ATP or adenosine, whereas the A1 receptor-selective antagonist DPCPX and the non-selective, membrane impermeable adenosine receptor antagonist 8-SPT

abolished the effects of adenosine, but only decreased those of ATP. The A2A receptor antagonistic CSC failed to interfere with any of the two agonists. A1 and P2Y1 receptors formed dimeric constructs in HEK293 cells, at which the affinity of DPCPX was reduced in a [3H]NECA radioligand binding assay, whereas the P2Y receptor agonist ADPb-S was capable of displacing the radioligand (Yoshioka et al., 2001). These findings were largely confirmed in/ex vivo, where the authors studied rat brain cortex, hippocampus and cerebellum as well as primary cultures of cortical neurons (Yoshioka et al., 2002). However, the existence of such a dimeric receptor was decided to be highly unlikely in the hippocampus, because the ATP-mediated inhibition of glutamate release was eliminated in brain slices from A1 knockout mice suggesting a direct effect at A1 receptors after rapid enzymatic breakdown (Masino et al., 2002). Thus, the hypothesis was forwarded that the inhibitory effects of ATP on hippocampal synaptic transmission require localized extracellular catabolism by ectonucleotidases and “channelling” of the generated adenosine to adenosine A1 receptors (Cunha et al., 1998). In the present study, a similar mechanism may operate in modulating the glutamatergic input onto MSNs. In fact, immunoisolated cholinergic nerve terminals of the rat striatum contained synaptic ectophosphohydrolases converting ATP to adenosine (Richardson et al., 1987), and ecto-ATPase and ecto-ATPdihydrolase were co-localized in caudate nucleus synaptic plasma

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Fig. 7. Effect of ATP on NMDA and AMPA currents evoked by rapid, local superfusion onto striatal medium spiny neurons. NMDA (100 mM) and AMPA (100 mM) were applied for 1e2 s every 200 s. (A) Original recordings of NMDA currents evoked before, during and after the application of ATP (300 mM; b) or without ATP (a). (Ac) The effect of ATP (300 mM) alone, as well as that of various antagonists on the EPSCs alone or in combination with ATP, is expressed as percentage inhibition. The time-dependent changes in NMDA currents are designated as controls. Ifenprodil (10 mM), PPADS (100 mM), and suramin (100 mM) by themselves depressed the NMDA currents, but did not interfere with the effect of ATP (300 mM). In contrast, DPCPX (0.3 mM), and PPADS (100 mM) plus DPCPX (0.3 mM) abolished the ATP (300 mM) effect. (Ad) ADP, AMP, adenosine (300 mM each), and MRS 2768 (100 mM) had no effect on the NMDA currents. CCPA (1 mM) facilitated, whereas CGS 21680 (0.1 mM) inhibited (although only in a subpopulation of neurons) the current caused by NMDA. The effect of CCPA was antagonized by MRS 1220 (10 mM), which by itself did not alter the NMDA currents. The ‘þ’ signs before ATP or CCPA signify that these agonists were applied in the presence of antagonists indicated below the preceding columns. Mean  S.E.M. of 6e14 cells. *P < 0.05; statistically significant difference from controls. **P < 0.05; statistically significant difference from the effect of ATP alone. (B) Original recordings of AMPA currents evoked before, during and after the application of ATP (300 mM; b) or without ATP (a). Representative experiment out of 5 similar ones.

membranes (Nedeljkovic et al., 2003). In addition, we proved that both the AMPA/kainate and NMDA components of the EPSC were depressed by ATP to a similar extent and DPCPX only moderately counteracted this effect. The residual action of ATP in the presence of DPCPX appeared to be, however, due to a blockade of postsynaptic NMDA receptors (abolished by a combination of DPCPX and AP-5) rather than postsynaptic AMPA/kainate receptors (not altered by a combination of DPCPX and NBQX). With these considerations in mind, we applied NMDA and AMPA onto MSNs to evoke transmembrane currents, and investigated the modulation of these responses by ATP. It was found that ATP depressed the NMDA-, but not the AMPA-induced currents. Ifenprodil, an antagonist of the NR2B subunit of NMDA receptors did not alter the inhibitory effect of ATP on the NMDA responses. This finding excludes an effect of ATP similar to that reported both for recombinant NMDA receptors expressed in oocytes and native recombinant NMDA receptors of rat cultured hippocampal neurons (Ortinau et al., 2003). These authors suggested that the molecular target of ATP is an adenine-nucleotide binding motif at the NR2B subunit; ATP was proposed to competitively interact with NMDA at this binding pocket. However, in our experiments, the findings that DPCPX, but not PPADS or suramin counteracted the postsynaptic effect of ATP indicated that A1 receptors are indirectly mediating the

ATP-induced inhibition of NMDA receptors. Thus, we conclude that only adenosine locally generated by the enzymatic degradation of ATP (“channelling” of adenosine to the synapse; Cunha et al.,1998) is able to interfere with NMDA. The depression of NMDA current responses by high concentrations of PPADS, suramin, and reactive blue-2, but not DPCPX, when given alone, may be due to the non-specific blockade of NMDA receptors as documented previously for rat locus coeruleus and mouse hippocampal neurons (Frohlich et al., 1996; Peoples and Li, 1998). Such a blockade of NMDA receptors by PPADS, suramin and RB-2 (RB-2 may block in addition also AMPA/kainate receptors and presynaptically inhibit the release of glutamate by blocking its vesicular uptake; Roseth et al., 1998), is suggested to be the reason for the time-dependent depression of EPSCs (see Fig. 3C). In contrast to the presence of A2A receptors in a subset of MSNs only, all afferent glutamatergic fibres projecting from the cortex and thalamus to the striatum appear to be endowed with A1 receptors. The present experiments give a reasonable explanation for the fact that ATP causes a higher maximum inhibition of the striatal EPSCs than adenosine, although only A1, but not P2Y receptor antagonists interfere with the inhibitory effect of both purine agonists. We suggest that adenosine acts at presynaptic A1 receptors only, whereas ATP, in addition of indirectly activating these receptors, also postsynaptically depresses the NMDA currents

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produced by nerve stimulation-evoked glutamate. It is an interesting observation that not ATP itself, but its enzymatic breakdown product adenosine interacted with NMDA at the postsynaptic site, whereas bath- or topically-applied adenosine was ineffective. Apparently this adenosine did not generate high enough local concentrations around the agonist recognition site of the postsynaptic A1 receptor negatively interacting with NMDA receptors. Acknowledgements This work was supported by a grant of the Deutsche Forschungsgemeinschaft, Bonn to PI and WN (IL 20/19-1) and a scholarship of the Cariplo Foundation to I.S. References Abbracchio, M.P., Burnstock, G., Boeynaems, J.M., Barnard, E.A., Boyer, J.L., Kennedy, C., Knight, G.E., Fumagalli, M., Gachet, C., Jacobson, K.A., Weisman, G.A., 2006. International Union of Pharmacology LVIII: update on the P2Y G protein-coupled nucleotide receptors: from molecular mechanisms and pathophysiology to therapy. Pharmacol. Rev. 58, 281e341. Amadio, S., Montilli, C., Picconi, B., Calabresi, P., Volonté, C., 2007. Mapping P2X and P2Y receptor proteins in striatum and substantia nigra: an immunohistological study. Purinergic Signal. 3, 389e398. Burnstock, G., 2007. Physiology and pathophysiology of purinergic neurotransmission. Physiol. Rev. 87, 659e797. Burnstock, G., Krügel, U., Abbracchio, M.P., Illes, P., 2011. Purinergic signalling: from normal behaviour to pathological brain function. Prog. Neurobiol. 95, 229e274. Calabresi, P., Mercuri, N., Stanzione, P., Stefani, A., Bernardi, G., 1987. Intracellular studies on the dopamine-induced firing inhibition of neostriatal neurons in vitro: evidence for D1 receptor involvement. Neuroscience 20, 757e771. Calabresi, P., Centonze, D., Pisani, A., Bernardi, G., 1997. Endogenous adenosine mediates the presynaptic inhibition induced by aglycemia at corticostriatal synapses. J. Neurosci. 17, 4509e4516. Centonze, D., Saulle, E., Pisani, A., Bernardi, G., Calabresi, P., 2001. Adenosinemediated inhibition of striatal GABAergic synaptic transmission during in vitro ischaemia. Brain 124, 1855e1865. Ciruela, F., Casado, V., Rodrigues, R.J., Lujan, R., Burgueno, J., Canals, M., Borycz, J., Rebola, N., Goldberg, S.R., Mallol, J., Cortes, A., Canela, E.I., Lopez-Gimenez, J.F., Milligan, G., Lluis, C., Cunha, R.A., Ferre, S., Franco, R., 2006. Presynaptic control of striatal glutamatergic neurotransmission by adenosine A1eA2A receptor heteromers. J. Neurosci. 26, 2080e2087. Cunha, R.A., Sebastiao, A.M., Ribeiro, J.A., 1998. Inhibition by ATP of hippocampal synaptic transmission requires localized extracellular catabolism by ectonucleotidases into adenosine and channeling to adenosine A1 receptors. J. Neurosci. 18, 1987e1995. Deussen, A., 2000. Metabolic flux rates of adenosine in the heart. Naunyn Schmiedebergs Arch. Pharmacol. 362, 351e363. Flagmeyer, I., Haas, H.L., Stevens, D.R., 1997. Adenosine A1 receptor-mediated depression of corticostriatal and thalamostriatal glutamatergic synaptic potentials in vitro. Brain Res. 778, 178e185. Fredholm, B.B., Dunwiddie, T.V., 1988. How does adenosine inhibit transmitter release? Trends Pharmacol. Sci. 9, 130e134. Fredholm, B.B., Abbracchio, M.P., Burnstock, G., Daly, J.W., Harden, T.K., Jacobson, K.A., Leff, P., Williams, M., 1994. Nomenclature and classification of purinoceptors. Pharmacol. Rev. 46, 143e156. Fredholm, B.B., IJzerman, A.P., Jacobson, K.A., Linden, J., Muller, C.E., 2011. International Union of Basic and Clinical Pharmacology. LXXXI. Nomenclature and classification of adenosine receptors e an update. Pharmacol. Rev. 63, 1e34. Frohlich, R., Boehm, S., Illes, P., 1996. Pharmacological characterization of P2 purinoceptor types in rat locus coeruleus neurons. Eur. J. Pharmacol. 315, 255e261. Illes, P., Alexandre, R.J., 2004. Molecular physiology of P2 receptors in the central nervous system. Eur. J. Pharmacol. 483, 5e17. James, S., Richardson, P.J., 1993. Production of adenosine from extracellular ATP at the striatal cholinergic synapse. J. Neurochem. 60, 219e227. Jarvis, M.F., Khakh, B.S., 2009. ATP-gated P2X cation-channels. Neuropharmacology 56, 208e215. Jenner, P., Mori, A., Hauser, R., Morelli, M., Fredholm, B.B., Chen, J.F., 2009. Adenosine, adenosine A 2A antagonists, and Parkinson’s disease. Parkinsonism Relat. Disord. 15, 406e413. Jiang, Z.G., North, R.A., 1991. Membrane properties and synaptic responses of rat striatal neurones in vitro. J. Physiol. 443, 533e553. Kawaguchi, Y., Wilson, C.J., Augood, S.J., Emson, P.C., 1995. Striatal interneurones: chemical, physiological and morphological characterization. Trends Neurosci. 18, 527e535. Khakh, B.S., Burnstock, G., Kennedy, C., King, B.F., North, R.A., Seguela, P., Voigt, M., Humphrey, P.P., 2001. International Union of Pharmacology. XXIV. Current status of the nomenclature and properties of P2X receptors and their subunits. Pharmacol. Rev. 53, 107e118.

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