Suppression of GABA input by A1 adenosine receptor activation in rat cerebellar granule cells

Neuroscience 162 (2009) 946 –958

SUPPRESSION OF GABA INPUT BY A1 ADENOSINE RECEPTOR ACTIVATION IN RAT CEREBELLAR GRANULE CELLS R. COURJARET,* M. TRÖGER AND J. W. DEITMER

P2X1-7 receptors have been identified in the cerebellar cortex, although the changes during development, the low level of protein expression and the specificity of antibodies have led to some contradictory results (Rubio and Soto, 2001; Sim et al., 2004; Hervàs et al., 2005; Xiang and Burnstock, 2005). However, to date, no direct electrophysiological evidence for in situ functional P2X receptors in the cerebellum has been reported, although these receptors may be involved in the modulation of the inhibitory input to Purkinje cells by ATP (Brockhaus et al., 2004). The metabotropic P2Y receptors are also present in the cerebellum, and a switch of the expression of P2Y1 from glia to neurons after the third postnatal week was recently proposed (Amadio et al., 2007). Functional investigation has shown that the intracellular calcium concentration in Bergmann glia, astrocytes and Purkinje cells was increased, and that the inhibitory synaptic input to Purkinje cells was modulated, following P2Y receptors activation (Kirischuk et al., 1995, 1996; Brockhaus et al., 2004). Expression of adenosine A1 receptors was also identified in the cerebellum, their major expression site being the parallel fibers terminals (Goodman and Snyder, 1982) the basket cells, and, to a minor extent, the granular cell layer (Rivkees et al., 1995). A well-documented function of A1 receptors in the cerebellar cortex is the attenuation of the evoked excitatory input to the Purkinje cell by decreasing presynaptic calcium influx in parallel fibers (Kocsis et al., 1984; Takahashi et al., 1995; Dittman and Regehr, 1996; Wall and Dale, 2007). In addition, several other targets of A1-receptor activation were suggested in the cerebellar cortex: modulation of the input from mossy fibers to granule cells (Maffei et al., 2002), and, on cultured Purkinje cells, reduction of mGluR1 signaling (Tabata et al., 2007). Finally, a recent report using multi-electrode recording from organotypic slice cultures revealed that PIA (R-phenylisopropyl-adenosine), an A1 receptor agonist, reduced the firing frequency of Purkinje cells (Kessler et al., 2008). Effects of purinergic receptor activation have been reported for granule cells in culture including promotion of cell death (Amadio et al., 2005), modulating intracellular calcium (Vacas et al., 2003), increasing Calcium-calmodulin Kinase II phosphorylation (León et al., 2006) and inducing glutamate release (León et al., 2008). In the present study, we have used the patch-clamp technique in the wholecell recording configuration to investigate the effects on purinergic modulation on the GABAergic input to granule cells in situ and its consequences on the membrane potential and cell excitability. Our results indicate that purinergic receptor agonists can induce a decrease of the GABAergic input to

Abteilung für Allgemeine Zoologie, Fachbereich Biologie, Universität Kaiserslautern, Postfach 3049, Erwin-Schrödinger-straße 13, D-67653, Kaiserslautern, Germany

Abstract—Synaptic transmission has been shown to be modulated by purinergic receptors. In the cerebellum, spontaneous inhibitory input to Purkinje neurons is enhanced by ATP via P2 receptors, while evoked excitatory input via the granule cell parallel fibers is reduced by presynaptic P1 (A1) adenosine receptors. We have now studied the modulation of the complex GABAergic input to granule cells by the purinergic receptor agonists ATP and adenosine in acute rat cerebellar tissue slices using the whole-cell patch-clamp technique. Our experiments indicate that ATP and adenosine substantially reduce the bicuculline- and gabazine-sensitive GABAergic input to granule cells. Both phasic and tonic inhibitory components were reduced leading to an increased excitability of granule cells. The effect of ATP and adenosine could be blocked by 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), but not by other P1 and P2 receptor antagonists, indicating that it was mediated by activation of A1 adenosine receptors. Our results suggest that, in the cerebellar network, A1 receptor activation, known to decrease the excitatory output of granule cells, also increases their excitability by reducing their complex GABAergic input. These findings extend our knowledge on purinergic receptors, mediating multiple modulations at both inhibitory and excitatory input and output sites in the cerebellar network. © 2009 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: cerebellar cortex, purinergic modulation, ATP, synaptic transmission, P1 receptors.

Purinergic signaling is now a well-recognized element of the CNS physiology, where ATP and its metabolites are involved in a wide range of processes. ATP alone for instance can act as a neurotransmitter, a cotransmitter and a neuromodulator (Burnstock, 2007). On the other hand, its metabolite, adenosine, has been identified as a presynaptic modulator regulating excitatory (Fredholm et al., 2005) and inhibitory neurotransmission (Chieng and Williams, 1998; Bagley et al., 1999; Shen and Johnson, 2003; Jeong et al., 2003; Yum et al., 2008). We have shown that purinergic receptors are expressed and functional in the cerebellum, mediating synaptic modulation (Brockhaus et al., 2004; Casel et al., 2005; Deitmer et al., 2006). All subtypes of the ionotropic *Corresponding author. Tel: ⫹49-0-631-205-2565; fax: ⫹49-0-631205-3515. E-mail address: [email protected] (R. Courjaret). Abbreviations: CPAdo, N6-cyclopentyladenosine; DMPX, 3,7-dimethyl1-propargylxanthine; DPCPX, 8-cyclopentyl-1,3-dipropylxanthine.

0306-4522/09 $ - see front matter © 2009 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2009.05.045

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granule cells via activation of adenosine A1 receptors which in turn can increase their excitability.

EXPERIMENTAL PROCEDURES Animals Wistar rats aged between 15 and 20 days old bred and housed in our local animal facility were used. The animal was decapitated and the cerebellum transferred in ice cold extracellular low calcium media of the following composition (in mM): NaCl 125, KCl 2.5, CaCl2 0.5, MgCl2 2.5, glucose 25, NaHCO3 26, NaHPO4 1.25, pH: 7.4, carbogen gassed (95% O2/5% CO2). Two hundred fifty micrometer thick parasagittal slices from the cerebellar vermis were cut using a Leica VTS-1000 vibratome (Leica, Wetzlar, Germany) and stabilized at 30 °C in the same saline during 1 h before experiments were carried out at room temperature (20 –24 °C). Animals were sacrificed in accordance with institutional animal care and use committee and animal welfare guidelines (Landesuntersuchungsamt Rheinland-Pfalz). Care was taken to minimize the suffering and the number of animals used.

Patch-clamp experiments The cerebellar slices were placed in a home-designed recording chamber and maintained under a nylon mesh held by a platinum wire and mounted on the fixed stage of a upright Axioskop microscope equipped with a 40⫻ lens (Zeiss, Oberkochen, Germany). They were continuously perfused with a gravity perfusion system that was also used to apply pharmacological compounds. The normal extracellular carbogen-gassed saline contained (in mM): NaCl 125, KCl 2.5, CaCl2 2, MgCl2 1, glucose 25, NaHCO3 26, NaHPO4 1.25, lactate 0.5, pH adjusted to 7.4 with NaOH. Patch pipettes were pulled from glass capillaries (GC150F10, Harvard Apparatus, Holliston, MA, USA) on a P-87 horizontal puller (Sutter Instruments, Novato, CA, USA) and fire polished. The resistance of the pipettes when filled with cesium-based solution was 5–7 M⍀, seals always had a resistance above 5 G⍀ and pipette capacitance was always compensated in the cellattached mode to ensure reliable reading of the cell capacitance values. For recording of synaptic events on granule cells, we increased the driving force for chloride and reduced K⫹ conductance using a CsCl-based intracellular solution containing (in mM): CsCl 125, EGTA 0.5, TEA-Cl 20, NaATP 2, NaGTP 0.5, MgCl2 2, Hepes 10, pH adjusted to 7.4 with CsOH. Signals were recorded using an HEKA EPC9 computer-driven amplifier associated with the Pulse software (ver. 8.8, HEKA, Lambrecht, Germany). Signals were filtered to 10 kHz and acquired at a 20 kHz sampling rate. The series resistance value was in the 20 – 40 M⍀ range, similar to previously described in the literature (Kaneda et al., 1995) and was not compensated. The change in series resistance was monitored during adenosine application and was non-significant (⫹1.5%⫾3.5%, n⫽10, P⫽0.67, ranging from ⫺6.6 to ⫹17.4%). Data analysis was performed using Clampfit 9.2 (Molecular Devices, Sunnyvale, CA, USA) and mini-analysis (Synaptosoft, Decatur, GA, USA). The event frequencies were averaged on periods of 60 s to perform statistical analysis. The measurement of the tonic noise was performed by averaging the standard deviation of the signal during 10 randomly selected “silent” (no synaptic events) periods of 100 ms. For current clamp experiments, a low chloride intracellular solution of the following composition was used: K-gluconate 140, EGTA 0.5, NaATP 2, NaGTP 0.5, MgCl2 2, Hepes 10, pH adjusted to 7.4 with KOH.

Pressure ejection GABA (10 and 100 ␮M) was pressure applied from an ejection pipette (patch pipette, 2.5 M⍀ resistance when filled with extra-

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cellular saline) positioned within 25 ␮m from the granule cell body and connected to a fast pressure ejection system (PDES02, NPI Electronics, Tamm, Germany) set to 1 bar. This technique allows very short applications as well as low dilution of the agonist reaching the cell (Di Angelantonio and Nistri, 2001).

Pharmacological agents All compounds were from Sigma Chemicals (Taufkirchen, Germany) except N-(2-methoxyphenyl)-N=-[2-(3-pyrindinyl)-4-quinazolinyl]-urea (VUF5574, Tocris, Ellisville, MO, USA). 8-Cyclopentyl-1, 3-dipropylxanthine (DPCPX), 3,7-dimethyl-1-propargylxanthine (DMPX), N6-cyclopentyladenosine (CPAdo) and VUF5574 were first dissolved in DMSO as stock solution, the final DMSO concentration never exceeded 0.1%.

Statistical analysis Data are given as means⫾standard error to the mean (SEM) of n number of cells. Significance was tested using repeated measure ANOVA followed by Newman–Keuls post-test and paired and unpaired Student’s t-test when applicable. Significance was ranked to three degrees: significant P⬍0.05 (*), very significant P⬍0.01 (**) and highly significant P⬍0.001 (***).

RESULTS Adenosine modulates the GABAergic input to granule cells Granule cells were identified according to their localization in the inner granular layer, their small cell body (⬇5 ␮m in diameter) and their small apparent capacitance (4.0⫾0.3 pF, n⫽9). Using a low chloride pipette solution (based on Kgluconate, see Experimental Procedures, ECl⫽⫺88.4 mV) synaptic events were recorded at depolarized membrane potential. In order to increase the driving force for chloride ions and to reduce the background noise, a CsCl-based intracellular solution was used subsequently (ECl⫽2.7 mV). This allowed stable recording of spontaneous synaptic events. The current–voltage relationship of these events was linear (r2⫽0.99⫾ 0.01, n⫽7), and the reversal potential was close to the chloride equilibrium potential (Erev⫽2.8⫾1.4 mV, n⫽7, not illustrated). Bath application of adenosine (100 ␮M, 3 min) induced a strong reduction of the synaptic event frequency from 2.4⫾0.4 to 0.8⫾0.3 Hz (n⫽17, P⬍0.001, Fig. 1a, d, f). This effect was also observed during application of the A1 receptor agonist CPAdo (10 ␮M, 1 min, Fig. 1b, g). However, contrary to the adenosine effect, the CPAdo-induced reduction of the synaptic activity was poorly reversible (two out of seven cells showed partial reversal). Adenosine in the extracellular compartment can originate from cells by being directly released, or by being the result of ATP release and subsequent degradation by ectonucleotidases (Casel et al., 2005; Fredholm et al., 2005; Wall and Dale, 2007). Accordingly, we tested the potency of ATP in mimicking the effect of adenosine. Bath application of ATP (100 ␮M, 3 min) also reduced the synaptic activity similar as adenosine (Fig. 1c, h). We performed a detailed analysis of the amplitudes and kinetics of the synaptic events before, during, and after purinergic agonist application (Fig. 1e). Except

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Fig. 1. Purinergic modulation of the synaptic input to granule cells. (a) Adenosine (ADO) reduced the frequency of synaptic events. The effect was reproduced using the A1 receptor agonist CPAdo (b) and during ATP application (c). (d) Adenosine induced no major modification of the events amplitude (see also Table 1). (e) Event kinetics before and during adenosine application. The traces represent 50 averaged currents in control condition and during adenosine application. The rise and decay phase were not affected by adenosine. (f– h) Histograms summarizing the effects of adenosine, CPAdo and ATP on the event frequency. Number of experiments are shown in the bars, statistics are given according to multiple measures ANOVA and Newman–Keuls post hoc test.

for a small but significant increase in the current amplitude induced by adenosine by 11.7%⫾3.8% (P⬍0.05), there was no detectable change in these parameters (Table 1). The tonic GABAergic input of granule cells is affected by adenosine Granule cells receive a complex GABAergic input. First, direct synaptic transmission and spillover of GABA beyond

the synaptic cleft create a two-component, fast and slow, respectively, phasic inhibitory response. Secondly, GABA induces a constant background chloride current producing a tonic inhibition of the neurons; this GABA is released by both vesicular and non-vesicular mechanisms, the relative contribution of both also depending on the age of the animal (Brickley et al., 1996; Wall and Usovicz, 1997; Hamann et al., 2002; Rossi et al., 2003; Farrant and Nusser, 2005). We have evaluated the relative contribution

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Table 1. Amplitude and kinetics of spontaneous inhibitory synaptic events recorded on granule cells before and during application of purinergic agonists

CTR ATP 100 ␮M P CTR ADO 100 ␮M P CTR CPAdo 10 ␮M P

Amplitude (pA)

Rise time(10–90%) (ms)

Decay ␶ (ms)

⫺37.7⫾3.3 ⫺38.7⫾(4)0.2 0.7438 (ns) ⫺40.6⫾2.6 ⫺45.1⫾3.1 mV 0.013 (*) ⫺44.0⫾7.1 ⫺41.0⫾7.1 0.479 (ns)

0.76⫾0.03 0.83⫾0.13 0.501 (ns) 0.73⫾0.03 0.72⫾0.03 0.545 (ns) 0.83⫾0.09 0.74⫾0.06 0.206 (ns)

14.8⫾1.1 14.8⫾1.1 0.960 (ns) 17.6⫾2.0 17.7⫾2.1 0.795 (ns) 23.6⫾5.4 24.3⫾5.0 0.794 (ns)

N 7 7 13 13 6 6

The decay ␶ was evaluated using a single exponential fit of the decay phase from 20% to 80% of the maximum current amplitude.

of both tonic and phasic input under our conditions using the two specific GABAA receptor antagonists bicuculline (10 ␮M) and gabazine (5 ␮M). Both compounds reversibly reduced the synaptic event frequency by more than 85%, illustrating that most, if not all, recorded events were GABAergic (Fig. 2a– c). Furthermore, a small upward deflection of the holding current was recorded following application of bicuculline (17.8⫾2.4 pA, n⫽13, P⬍0.0001) and gabazine (16.6⫾4.3 pA, n⫽4, P⬍0.05, Fig. 2a, b). This sustained net current represents the blocking of the tonic current by GABA antagonists on granule cells (Brickley et al., 1996; Rossi et al., 2003; Chadderton et al., 2004). The amplitude of this current was measured by averaging the baseline level on 10 periods of 100 ms, a method that does not allow detection of very small variations in the background current (Glykys and Mody, 2007) and is very sensitive to any change in the holding current. As illustrated in Fig. 2b, the inhibition of the background current is associated with a decrease in the background noise. An alternative method to evaluate this resting current is to measure the standard deviation of the current baseline (Caraiscos et al., 2004; Park et al., 2006). This was performed by averaging the standard deviation of the signal in 10 “silent” periods (no synaptic events) of 100 ms. As indicated in Fig. 2d, the standard deviation was significantly reduced following application of the GABA receptors antagonists (⫺44.7%⫾3.8% for bicuculline, n⫽13, P⬍0.001, and ⫺42.4%⫾1.6%, n⫽4, P⬍0.01 for gabazine). Moreover, the standard deviation value followed the chloride gradient, decreasing from 5.8⫾0.5 pA at ⫺90 mV to 3.0⫾0.1 pA at a holding potential of ⫹10 mV, and increasing for more depolarizing potentials (n⫽6, not illustrated). Thus, under our experimental conditions, we were able to measure both “phasic” and “tonic” GABAergic input of granule cells. A similar analysis was also performed on the tonic noise recorded before, during and after application of adenosine, CPAdo and ATP. During purinergic agonist application, small positive deflections of the current baseline were detected (4.8⫾1.7 pA, n⫽23, P⬍0.01 for 100 ␮M adenosine, 6.6⫾2.2 pA, n⫽5, P⬍0.05 for 10 ␮M CPAdo; 3.5⫾1.2 pA, n⫽17, P⬍0.01 for 100 ␮M ATP). To confirm the modulation of the resting current, we measured its

standard deviation before, during, and after application of the purinergic agonists. As illustrated in Fig. 3a– d, all three compounds induced a small but significant reduction of tonic noise (⫺10.7%⫾ 2.1% for adenosine, n⫽24, P⬍0.001; ⫺10.8%⫾1.9%, n⫽7, P⬍0.05 for CPAdo and ⫺13.2%⫾2.4%, n⫽17, P⬍0.001 for ATP). Similar to the modulation of synaptic event frequency, the effect of CPAdo on tonic noise could not be reversed. Experiments were then performed to estimate the contribution of the GABAergic input to the fraction of the tonic input affected by adenosine. During gabazine application (5 ␮M), adenosine failed to reduce the standard deviation of the current baseline (Fig. 3e). Finally, since the TTX-sensitive fraction of the tonic current component depends on the age of the animal (Wall and Usowicz, 1997), we evaluated its contribution to the adenosine-sensitive tonic noise. Bath application of TTX alone (0.5 ␮M) produced a reduction of the tonic noise (⫺14.4%⫾2.1%, n⫽6, P⬍0.05, not illustrated) and no further reduction was obtained when adenosine was applied in the presence of TTX (P⫽0.88). We conclude that adenosine receptor activation is able to inhibit both the phasic and the tonic GABAergic input to granule cells presumably through a reduction of action potential– dependent events. Adenosine fails to modulate GABA currents induced by pressure application The absence of important modifications in the amplitude and kinetics of GABAergic synaptic events following adenosine application suggested a predominantly presynaptic action. To rule out a possible postsynaptic effect, we evaluated the influence of adenosine on ionic currents induced by focal pressure application of GABA onto granule cells. Application of GABA from a pipette containing 100 or 10 ␮M (duration: 10 ms, pressure: 1 bar, Fig. 4a, c) at a holding potential of ⫺70 mV (CsClbased intracellular solution), induced bicuculline-sensitive transient inward currents (Fig. 4). Both signals evoked by 100 and 10 ␮M GABA were unaffected by bath application of adenosine (100 ␮M, Fig. 4). The decay kinetics of the currents were also unaffected (Fig.

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Fig. 2. Characteristics of the synaptic input to granule cells. (a, c) The event frequency was strongly and reversibly reduced by the GABAA receptor antagonists gabazine (Gbz and bicuculline (Bic). (b) The inhibition by Bic of GABAA receptors unmasked the tonic inhibition of granule cells. The holding current is reduced as well as the background noise. (d) The measure of the standard deviation allowed quantification of the reduction in the background current induced by complete inhibition of GABAA receptors by Gbz and Bic.

4a, c). This is in line with the suggestion that the main site of action of adenosine is presynaptic. Adenosine activates an A1 receptor subtype To further identify the target of adenosine, we evaluated the efficacy of adenosine receptor-specific inhibitors (Muller et al., 1997; Baraldi and Porea, 2000; Burnstock, 2007). Among adenosine receptor subtypes, the A1 receptor seems to predominate in the cerebellum (Fredholm et al., 2005) with higher expression in the molecular layer as compared to the granular layer (Goodman and Snyder, 1982; Rivkees et al., 1995). Since the A1 receptor agonist CPAdo reproduced the adenosine effect, we first tested

DPCPX, an A1 receptor antagonist. Application of DPCPX (2 ␮M) alone led to no significant change (Fig. 5b) in the synaptic event frequency suggesting little or no tonic activation of A1 receptors under control conditions. The decline of the frequency of synaptic events induced by adenosine (100 ␮M) was reduced from ⫺69.2⫾5.8% to ⫺20.6⫾4.6% (n⫽10, P⬍0.001, Fig. 5a, b) during application of 2 ␮M DPCPX. As observed for the transient synaptic events, DPCPX (2 ␮M) alone had no influence, but fully reversed the adenosine (100 ␮M) effect on the tonic noise (Fig. 5c). To rule out the contribution of other adenosine receptor subtypes, we repeated these experiments using a mixture of the A2 receptor antagonist DMPX and of

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Fig. 3. Purinergic receptor activation reduced the tonic GABA input on granule cells. Cells were voltage clamped at ⫺70 mV. (a) Current traces illustrating the reduction in the tonic noise of the recording during adenosine application. (b– d) The standard deviation of the current was reduced during application of adenosine (b), the A1 receptor agonist CPAdo (c) and ATP (d). (e) During blockade of the GABA input by gabazine, ADO did not further affect the standard deviation of the current.

the A3-antagonist VUF 5574 (N-(2-methoxyphenyl)-N=[2-(3-pyrindinyl)-4-quinazolinyl]-urea, 1 ␮M). These two compounds had no significant effect on the adenosineinduced modulation of the synaptic event frequency (not illustrated).

We next evaluated, if the effect of ATP on the synaptic events was solely caused by its degradation to adenosine, activating A1 receptors. First, we excluded the contribution of P2 purinergic receptors using the broad-spectrum antagonist PPADS (Ralevic and Burnstock, 1998; Brockhaus

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Fig. 4. Adenosine failed to modulate currents elicited by focal application of GABA. The pressure ejection of 100 ␮M (a, b) and 10 ␮M (c, d) GABA onto a granule cell induced transient bicuculline-sensitive inward currents (ECl ⬃3 mV). For both GABA concentrations tested, adenosine did affect neither current amplitude nor kinetics.

et al., 2004). Although PPADS (10 ␮M) did promote an increase in the frequency of synaptic events, it had no blocking effect on the ATP-induced reduction of the event frequency (not illustrated). Conversely, DPCPX (2 ␮M) completely inhibited the effect of ATP on the GABAergic events (Fig. 6a, b). In a recent report it has been estimated that perfusion of 100 ␮M ATP on a cerebellar slice led to the production of 3.8 ␮M adenosine by enzymatic degradation (Wall et al., 2007). The effect of 100 ␮M ATP on synaptic event frequency being similar in amplitude to 100 ␮M adenosine (respectively ⫺85.8⫾6.4, n⫽10 and ⫺71.7%⫾

4.8%, n⫽17, P⫽0.24), we therefore repeated the experiments using a lower adenosine concentration (5 ␮M). Under these conditions, 2 ␮M DPCPX completely inhibited the adenosine effect on the synaptic events (Fig. 6c). However, the effect of 5 ␮M adenosine was smaller (⫺38.8%⫾5.6%, n⫽7) than the effect of 100 ␮M ATP (⫺85.8%⫾6.4%, n⫽10, P⬍0.001) suggesting that under our conditions a higher concentration of adenosine might be produced during the application of 100 ␮M ATP, and/or that removal of adenosine from the extracellular space effectively lowers the adenosine concentration.

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2005). ARL67156 alone had no significant effect on the synaptic event frequency but reduced the effect of ATP (from ⫺58.8⫾11.1% to ⫺32.2⫾9.6%, n⫽5, P⬍0.05, not illustrated). Taken together, these results demonstrate that adenosine reduces both the phasic and the tonic inhibitory input to granule cells via activation of the A1 receptor subtype.

Fig. 5. Adenosine activated an A1 receptor. (a) The inhibition of synaptic event frequency induced by adenosine was reduced during application of the A1 antagonist DPCPX. (b) Histogram summarizing the effect of adenosine and DPCPX on the synaptic event frequency. (c) DPCPX inhibited the reduction of the tonic noise induced by adenosine. DPCPX alone had no effect on either the standard deviation of the current or the synaptic event frequency.

Finally, we examined if inhibiting the ectonucleotidases involved in the degradation of ATP to adenosine had an effect on the ATP-induced modulation of the synaptic input. We applied ATP together with 50 ␮M ARL67156 (Zimmermann, 2000) whose potency in inhibiting ATP degradation has been shown in the cerebellum (Casel et al.,

Fig. 6. ATP mimicked the effects of adenosine on granule cells. (a) ATP-induced reduction of the synaptic event frequency was blocked by the A1 antagonist DPCPX. (b) Histogram summarizing the effect of ATP and DPCPX on the synaptic event frequency. (c) Adenosine applied to a concentration of 5 ␮M (matching the concentration suspected to be produced by ATP degradation) did not fully mimic the inhibition of GABAergic events induced by ATP. Two micromolar DPCPX fully inhibited the effect of 5 ␮M adenosine.

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effect induced by adenosine (100 ␮M) on the resting membrane potential was similar to the depolarization induced by bicuculline (10 ␮M) in the same recording conditions (3.6⫾2.0 mV, n⫽7 for adenosine vs. 8.9⫾3.0 mV, n⫽19 for bicuculline, P⫽0.31). On the other hand, during the application of ATP (100 ␮M, 3 min), several larger depolarizing events were recorded. The average peak amplitude of these events (maximum depolarization recorded on each cell) was above 30 mV (Fig. 7a, b), triggering action potential(s) in three out of seven cells tested. The striking difference between the effects of adenosine and ATP suggested a yet unknown, additional, effect of ATP that promotes the depolarization of the granule cells. Although the blocking of the GABA input to granule cells alone does

Adenosine increases the excitability of granule cells Granule cells fire action potentials following summation of two or more EPSCs, and one mossy fiber presynaptic volley might be sufficient to produce these synaptic events (Chadderton et al., 2004; Rancz et al., 2007). The excitability of granule cells is greatly influenced by their complex GABAergic input, and tonic inhibition is crucially involved in the regulation of excitability (Hamann et al., 2002; Farrant and Nusser, 2005). Experiments were performed in current clamp conditions to evaluate the ability of adenosine to depolarize or to increase the excitability of granule cells. As illustrated in Fig. 7a, b, bath application of adenosine (100 ␮M, 3 min) only induced small or no depolarization of the resting membrane potential. The average

b ADO 100 µM

-50

-100

0

5

10 15 Time (min)

20

50 **

ATP 100 µM

Depolarization (mV)

Membrane potential (mV)

a 0

25

40 30 20 ns

10 0

n=7

d

c

ADO 100 µM

Current injection (pA)

20 mV 100 ms

8 pA

10 pA

ns

7.5 5.0 2.5 0.0

6 pA

n=7

***

10.0

Ctr

n=19

Bic ADO ATP 100 µM 10 µM 100 µM

n=16

Ctr

n=11

ADO Bic Bic 100 µM 10 µM 10 µM + ADO 100 µM

Fig. 7. Purinergic modulation of the granule cell excitability. (a) Adenosine induced no or a very small depolarization of the granule cells. Conversely, ATP induced large depolarizing events. (b) Histogram summarizing the effects of adenosine, ATP and bicuculline (Bic) on the membrane potential. The average effect of adenosine was similar to Bic. (c) The excitability of granule cells was evaluated by injecting depolarizing current pulses (2 pA increment, 200 ms in duration, 1 Hz). Adenosine augmented the number of triggered spikes for a given current intensity indicating increased excitability of granule cells in the presence of adenosine. (d) Adenosine lowered the threshold current. This effect was absent when Bic was added prior to adenosine.

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apparently not promote a strong depolarization, an increase in excitability has been reported both in situ and in vivo when GABA antagonists are applied (Brickley et al., 1996; Hamann et al., 2002; Chadderton et al., 2004). We evaluated the effect of adenosine on the excitability of granule cells by measuring the efficacy of depolarizing current pulses to trigger action potentials (2 pA increment, 200 ms, 1 Hz, from ⫺20 to ⫹20 pA, Fig. 7c, d). The granule cells investigated had an average membrane potential of ⫺70.0 mV⫾2.3 (n⫽31) and a membrane resistance ranging from 0.7 to 4.6 G⍀ (measured for a ⫺10 pA current step, mean value 2.2⫾0.2 G⍀, n⫽31). Among all cells investigated, 16 out of 31 did show an adenosine-induced (100 ␮M) increase in excitability, the effect being reversible in six of them. In these cells, the amount of current required to trigger the first action potential decreased during adenosine application from 8.2⫾1.6 to 5.9⫾1.5 pA (n⫽16; P⬍0.001, Fig. 7c, d). This effect was not recorded when bicuculline (10 ␮M) was added prior to adenosine application (Fig. 7d) the maximum firing frequency of the cell was, however, not affected by adenosine application (70.5⫾8.5 Hz under control conditions vs. 74.1⫾7.5 Hz in adenosine, n⫽16, P⫽0.19). In cells where an increased excitability was detected, the membrane resistance only changed from 2.3.⫾0.3 to 2.4⫾0.4 G⍀ (n⫽16, P⬍0.05, for a ⫺10 pA current step). This very small change in resistance could explain that the effect of adenosine on excitability was only recorded in 50% of the cells investigated. Taken together, these results show that adenosine could contribute to the regulation of the granule cell excitability.

DISCUSSION We have investigated the action of purinergic receptor activation on granule cells of the cerebellar cortex in juvenile rats. Analysis of their complex inhibitory input revealed that adenosine activated A1 receptors which promote a reduction of the phasic and tonic GABAergic inhibition. Current clamp experiments indicated that this adenosineinduced decrease of the GABAergic input to granule cells produced only a small depolarization of the neuron, but that the reduction of the chloride shunting inhibition of the cells was able to increase their excitability. A1 receptor activation downregulates GABAergic transmission Activation of A1 receptors is generally associated in the brain with a reduction in neurotransmitter release, an effect mainly described for excitatory synapses (Fredholm et al., 2005). Nevertheless, the presynaptic downregulation of GABAergic neurotransmission has also been reported in several preparations such as immature hippocampal CA1 neurons (Jeong et al., 2003), tuberomammillary nucleus neurons (Yum et al., 2008), periaqueductal grey neurons (Bagley et al., 1999) and nucleus accumbens (Chieng and Williams, 1998). In all these cases it has been shown that the A1 receptor-mediated modulation was associated with the regulation of cAMP-dependent signaling pathways. The

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precise target of the modulation is not known yet, but the modulation of inwardly rectifying potassium and calcium channels has been suggested (Jeong et al., 2003; Yum et al., 2008). In the present work, evidence suggests that the target of adenosine was presynaptic: (1) the modulation of the synaptic events induced by adenosine did not affect their kinetics and had only a minor effect on their amplitude; (2) adenosine failed to modulate currents induced by focal application of GABA onto the granule cells; (3) adenosine did not affect the tonic GABAergic current during TTX application. This suggests that the effect of adenosine is mainly due to an inhibition following A1 receptor activation of the action potential– driven GABA release responsible for both phasic synaptic events and accounting for the TTX-sensitive fraction of the background chloride current. Localization of A1 receptors in the granular layer The detection of A1 receptors in the cerebellum using either immunohistological techniques or autoradiography indicated expression in the whole cerebellum of both rats and mice predominantly in the molecular layer (Goodman and Snyder, 1982; Goodman et al., 1983; Rivkees et al., 1995; Laitinen, 1999). This pattern is likely to illustrate the expression of the receptors on the axon terminals of granule cells (i.e. parallel fibers), since the detection of adenosine receptors was strongly reduced in the “Weaver” mutant mice lacking the granule cells (Goodman et al., 1983). In addition, the modulation of these terminals by adenosine has been previously reported (Kocsis et al., 1984; Takahashi et al., 1995; Dittman and Regehr, 1996). The Golgi cells, that provide the main inhibitory synaptic input to granule cells, do not seem to express A1 receptor (Rivkees et al., 1995). An alternative site of action for adenosine could therefore be the terminals of the parallel fibers that feed back onto Golgi cells and regulate their firing. However, this is unlikely to be a major target of adenosine under our experimental conditions, since adenosine receptor activation is able to block almost completely the synaptic GABAergic input, while the modulation of Golgi cells by parallel fibers has a rather low efficacy (Dieudonne, 1998). Another putative localization of the receptor could be the axon terminals of the Golgi cells forming inhibitory synapses with the granule cells. The relatively weak expression of the A1 receptor in the granular layer, as reported by several groups, however, has so far occluded their precise location (Goodman and Snyder, 1982; Goodman et al., 1983; Rivkees et al., 1995; Laitinen, 1999). Further experiments are required to define more precisely the site of expression of adenosine receptors in the granule cell layer of the cerebellum. These include co-staining of A1-receptors and Golgi cells, analysis of the effect of adenosine on the paired-pulse ratio of GABAergic currents onto granule cells induced by electrical stimulation in the granular layer, and evaluation of the putative action of adenosine directly onto Golgi cells.

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Contribution to the control of excitability of granule cells The significance of the background inhibition on granule cell excitability has been well characterized in situ and in vivo (Brickley et al., 1996; Hamann et al., 2002; Chadderton et al., 2004). In cerebellar slices, removal of tonic inhibition using bicuculline or the ␣6-containing GABA receptor-selective antagonist furosemide reduced the amount of current needed to initiate action potential firing and increased their firing rate (Brickley et al., 1996; Hamann et al., 2002). In vivo, summation of excitatory postsynaptic potentials from mossy fibers (EPSPs) was required to trigger action potentials, while in the presence of gabazine, a single EPSP was sufficient (Chadderton et al., 2004). Our results clearly indicate that tonic and phasic GABAergic input to the granule cells was reduced by the activation of adenosine A1 receptors, and that this likely contributes to the increased excitability of the cell. Under our experimental conditions, the phasic inhibition contributed only to a small fraction of the GABA-induced charge transfer (11.4%⫾3.1%, n⫽12), similar to the value (under 10%) previously reported for p18 animals (Brickley et al., 1996), showing that the background current was prevalent in the GABA-induced modulation of excitability. Accordingly, the selective blockade of ␣6-containing GABA receptors by furosemide was shown to be sufficient to decrease the amount of current injection required for spiking by 39% (Hamann et al., 2002). Under our conditions, adenosine produced a small reduction of the tonic current (⫺10.7%⫾ 2.1% decrease of the SD) compared to that observed during bicuculline application (⫺44.7%⫾3.8%). This is in line with our finding that the increase in excitability induced by adenosine was limited and could not be recorded in all cells investigated. Several reports have indicated that the efficacy of stimulating parallel fibers was reduced following A1-receptor activation (Kocsis et al., 1984; Takahashi et al., 1995; Dittman and Regehr, 1996). We have confirmed that adenosine reduced the postsynaptic responses in voltageclamped Purkinje neurons evoked by parallel fiber stimulation under our conditions (Casel, 2006). Moreover, modulation of the input from mossy fibers to the granule cells by adenosine receptor activation has also been reported (Maffei et al., 2002). Hence, the granule cell/parallel fiber activity as modulated via adenosine A1 receptors seems to be tuned both at the somatic/dendritic excitatory and inhibitory input site and at the axonal/terminal output site. This complex balance might explain why bath-applied adenosine fails to modulate spontaneous synaptic input to Purkinje cells originating directly from parallel fibers (Dittman and Regehr, 1996), or indirectly from molecular layer interneurons, as both are under the control of parallel fiber activity (Casel et al., 2005). Adenosine release in the cerebellar cortex? Several sources of extracellular adenosine have been suggested in the brain (Fredholm, 2005). In the cerebellum, at least two different possible mechanisms can be proposed.

First, direct, action potential– dependent release of adenosine by axon terminals has been suggested for parallel fibers (Wall and Dale, 2007), and secondly degradation of extracellular ATP to adenosine following release of ATP has been suggested. Ectonucleotidases are active in the cerebellar slice and can produce adenosine in the micromolar range by breaking down ATP (Casel et al., 2005; Wall et al., 2007). Furthermore, ATP and adenosine levels have been revealed following blockade of either A1 (Takahashi et al., 1995; Laitinen, 1999; Savinainen, 2003) or P2 receptors (Brockhaus et al., 2004). The origin of extracellular ATP is not yet defined, but ATP might be released from astrocytes (Fields and Burstock, 2006), parallel fibers terminals (Wall and Dale, 2007; Beierlein and Regehr, 2006), or inhibitory interneurons in the molecular layer (Piet and Jahr, 2007) and then be degraded to adenosine. Unfortunately, the determination of basal and triggered concentrations of ATP and adenosine in the cerebellar slice is still difficult, because of the low purine levels and/or their fast degradation (Wall et al., 2007). Nevertheless, the adenosine tone does not seem to be sufficient under our recording conditions to produce a detectable reduction of the synaptic events in granule cells, since DPCPX application alone, to block adenosine A1 receptors, had no significant effect on the GABAergic input to granule cells. It should be noted that experimental parameters, such as O2 concentration, can strongly affect or even be responsible for the adenosine tone in brain slices (see Gordon et al., 2008). Moreover, since there are two different sites at which adenosine affects the same cell depending on the compartment (somato-dendritic or axonal), the adenosineinduced modulation would also depend on the site of origin of adenosine in the molecular or in the granular layer. Finally, we did show that ATP had a larger depolarizing effect than adenosine through a yet unknown mode of action. Therefore, if adenosine modulating the GABAergic input to granule cells originates from the degradation of ATP, the activity and location of ectonucleotidases would also be key factors determining the action of purines onto granule cells. Acknowledgments—This work was supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 530, Teilprojekt B1).

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(Accepted 21 May 2009) (Available online 27 May 2009)