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Pharmacologie de la Synapse, Institut de Biochimie et de Biophysique Moléculaire et Cellulaire, Université Paris-Sud, Orsay Cedex, France
Submitted 2 April 2007; accepted in final form 7 September 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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-benzyloxyaspartate (D-TBOA) and blocked by the desensitizing kainate (KA) receptors agonist SYM 2081, by nonsaturating concentrations of 6-cyano-7-nitroquinoxaline-2-3-dione (CNQX) [but not by GYKI 52466 hydrochloride (GYKI)] and by dialyzing PCs with guanosine 5'-[-thio]diphosphate (GDP-S). An endocannnabinoid-independent suppression of PF-EPSCs was also present in nearly mature wild-type mice but was absent in GluR6–/– mice. The endocannnabinoid-independent suppression of PF-EPSCs induced by mGluR1 agonists and the KA-dependent component of depolarization-induced suppression of excitation (DSE) were blocked by ryanodine acting at a presynaptic level. We conclude that retrograde release of Glu by PCs participates in mGluR1 agonist-induced suppression of PF-EPSCs at nearly mature PF-PC synapses and that Glu operates through activation of presynaptic KA receptors located on PFs and prolonged release of calcium from presynaptic internal calcium stores. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Levenes et al. 2001; Maejima et al. 2001) or by sustained parallel fiber (PF) stimulation (Brown et al. 2003; Maejima et al. 2001; Neale et al. 2001) inhibits both excitatory and inhibitory inputs to these neurons by retrograde signaling (Brown et al. 2003; Galante and Diana 2004; Levenes et al. 2001; Maejima et al. 2001; Marcaggi and Attwell 2005). Endocannabinoids have been favored as the retrograde messenger involved in this signaling (Galante and Diana 2004; Maejima et al. 2001), as was previously shown for depolarization-induced suppression of inhibition (DSI) (Diana et al. 2002; Glitsch et al. 1996, 2000; Kreitzer and Regehr 2001a; Llano et al. 1991a; Ohno-Shosaku et al. 2001; Pitler and Alger 1992, 1994; Vincent et al. 1992; Wang and Zucker 2001; Wilson and Nicoll 2001; Wilson et al. 2001) and for depolarization-induced suppression of excitation (DSE) (Brenowitz and Regehr 2003; Kreitzer and Regehr 2001b; Safo and Regehr 2005). However, the study by Levenes et al. (2001) contrasts with these results, suggesting instead that retrograde release of glutamate (Glu) by PCs was responsible for the observed agonist-dependent suppression of PF-excitatory postsynaptic currents (EPSCs), through activation of presynaptic ionotropic Glu receptors borne by PFs. Because most studies on the role of endocannabinoids in retrograde signaling at PF-PC synapses have been performed in juvenile rats and mice (but see Safo and Regehr 2005), whereas the study by Levenes et al. (2001) was performed in nearly mature rats, the apparent discrepancy on the nature of retrograde messengers involved in agonist-induced suppression of excitation at PF-PC synapses might be related to developmental differences. Indeed, we now know that DSE at PF-PC synapses is entirely mediated through retrograde release of endocannabinoids in juvenile rodents, whereas it also involves retrograde release of Glu in nearly mature animals (Crepel 2007).
Therefore these experiments were designed to determine whether such distinct mechanisms are also involved in suppression of PF-EPSCs by activation of postsynaptic mGluR1 in juvenile and nearly mature rats and mice. Because presynaptic kainate (KA) receptors are involved in DSE in nearly mature PF-PC synapses (Crepel 2007), emphasis was made on a possible role of these receptors in agonist-induced suppression of PF-EPSCs in nearly mature PF-PC synapses, as well as on a possible participation of presynaptic calcium-induced calcium release in this process.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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1 h. The recording chamber was perfused at a rate of 2 ml/min with oxygenated saline solution containing (in mM) 124 NaCl, 3 KCl, 24 NaHCO3, 1.15 KH2PO4, 1.15 MgSO4, 2 CaCl2, 10 glucose, and the GABAA antagonist bicuculline methochloride (10 µM, Sigma Aldrich, St. Quentin Fallavier, France), osmolarity 320 mOsm, final pH 7.35 at 27–28°C except when otherwise specified. PCs were directly visualized with Nomarski optics through a x40 water-immersion objective of an upright microscope (Zeiss).
Drugs were added to the superfusate. (S)-3,5-dihydroxyphenylglycine (DHPG), domoate, D-threo--benzyloxyaspartate (D-TBOA), 6-cyano7-nitroquinoxaline-2-3-dione (CNQX), GYKI 52466 hydrochloride (GYKI), D-2-amino-5-phosphopentanoic acid (D-APV), (2S,4R)-4methylglutamic acid (SYM 2081), 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), CGP55845-A,N-(piperidin-1-yl)-5-(4-iodophenyl)-1(2,4dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM-251), and ryanodine were purchased from Tocris (Illkirch, France). The CB1 cannabinoid receptor antagonist SR141716-A [N-(piperidin-1-yl)-5- (-4chlorophenyl)-1-(2.4-dichlorophenyl)-4-methyl-1H-pyrazol-3-carboxamide hydrochloride] was provided by Sanofi-Recherche (Montpellier, France). NG-nitro-L-argininemethyl ester (L-NAME) and guanosine 5'-[-this]diphosphate (GDP-S) was purchased from Sigma Aldrich. Stock solutions of drugs (dissolved in water or DMSO depending on manufacturer recommendations) were added to the oxygenated saline solution at the desired concentration.
Electrophysiology
Whole cell patch-clamp recordings were performed from PC somas, using an Axopatch-200A amplifier (Axon Instruments). Stimulating electrodes consisted in saline filled monopolar electrodes. PF stimulations were performed at 0.33 Hz, except for studies on unitary quantal events and on DSE, where stimulation rates were 0.1 and 0.5 Hz, respectively. Patch pipettes (2–4 M
) were filled with a solution containing (in mM) 140 K-gluconate, 6 KCl, 10 HEPES, 0.75 EGTA, 1 MgCl2, 4 Na2-ATP, and 0.4 Na-GTP, pH 7.35 with KOH; 300 mOsm. In experiments on DSE, patch pipettes (2–4 M) were filled with a solution containing (in mM) 140 Cs-gluconate, 6 KCl, 10 HEPES, 0.2 EGTA, 1 MgCl2, 4 Na2-ATP, and 0.4 Na-GTP (pH and osmolarity adjusted accordingly). The components of internal solutions were purchased from Sigma Aldrich.
In the cells retained for analysis, access resistance (usually 5–10 M) was partially compensated (50–70%), according to the procedure described by Llano et al. (1991b). Cells were held at a membrane potential of –70 mV, and PF-EPSCs were subjected to 10-mV hyperpolarizing voltage steps that allowed monitoring of the passive electrical properties of the recorded cell throughout the experiment (Llano et al. 1991b).
For paired-pulse facilitation (PPF) experiments (Atluri and Regehr 1996; McNaughton 1982; Schultz et al. 1994), PF stimulations of the same intensity were applied to the cell with an interstimulus interval of 30 ms, and the ratio of the amplitude of the second PF-EPSC over the first one was calculated on-line. Mean PPF values were obtained by averaging PPFs in individual traces for each cell studied in a given condition. Although this conventional method can produce spurious results (Kim and Alger 2001), no use was made of the alternative method proposed by these authors because both methods produced very similar results in a recent study on DSE at PF-PC synapses (Crepel 2007). Because PPF increases associated to agonist-induced suppression of PF-EPSCs could be small in certain experimental conditions (see RESULTS) and obscured by variability of basal PPF across cells, mean PPFs were further normalized in these experiments by determining for each cell mean normalized PPF = 100 x (mean PPF/PPFi), where PPFi was the individual PPF value of the last trace preceding the depolarizing step. The contribution of presynaptic factors in the variation of synaptic responses was also examined by using the coefficient of variation (CV) (Kullmann 1994; Martin 1966), where CV is given by: CV2 = (s/M)2, in which s is the SD of the amplitude distribution of EPSCs corrected for the background noise, and M is the mean amplitude of EPSCs during the same epoch. In these experiments, CV were calculated on sets of 40–60 stable EPSCs according to the procedure previously described (Blond et al. 1997).
Fluorometry
Parallel fibers in coronal slices from nearly mature and juvenile rats (see RESULTS) were loaded by focal application of 100 µM of the low-affinity calcium sensitive dye Fluo-4FF-AM (Molecular Probes) as previously described (Levenes et al. 2001). After loading for 45 min, fluorescent signals from labeled PFs were recorded in a 20 x 50-µm window placed above the molecular layer, 500–800 µm away from the loading site and 100 µm above PC layer. The epifluorescence excitation light at 485 ± 22 nm was gated with an electromechanical shutter (Uniblitz, Rochester, NY), and the emitted light was collected by a photometer through a barrier filter at 530 ± 30 nm. The rather selective and weakly desensitizing KA receptor agonist domoate (Lerma et al. 1993) was applied for 1 min and at a concentration of 20 µM by local superperfusion through a theta-tube (rate of 0.5 ml/min) placed 
50 µm above the surface of the slice, at the level of the molecular layer and parallel to labeled PFs. With such a protocol that minimized possible movements of the slice when switching superfusion through the theta-tube from standard saline solution to that with domoate included, this compound was unlikely to diffuse in significant concentration to nearby granule cells, whereas it was probably still at near saturing concentrations for presynaptic KA receptors (Renard et al. 1995), and this, even after partial dilution during superfusion of the tissue. In these experiments, the 1-min superfusion duration was chosen because, in pilot experiments, it was the shortest time that gave rise to reliable presynaptic calcium signals. Fluorescence signals corrected for dye bleaching and background fluorescence were expressed as relative fluorescence changes 
F/F, where F was the baseline fluorescence intensity and F was the change induced by domoate.
In sagittal slices, epifluorescence microscopy was also used to detect variations in intracellular free calcium concentration changes from an area of 90 x 90 µm centered on dendrites of recorded PCs, as previously described (Crepel 2007). In these experiments, the low affinity and impermeant calcium indicator Fluo-4FF (100 µM, Molecular Probes) was added to the same Cs-gluconate–based solution as used in experiments on DSE. The recording session started 30–45 min after whole cell break in, to allow diffusion of the dye to the dendrites. Fluorescence data corrected for dye bleaching and background fluorescence were again expressed as changes in F/F, where F was the baseline fluorescence intensity, and F was the fluorescence change induced by depolarization of PC soma from –70 to 0 mV for 1 s.
In all cases, pre- and postsynaptic calcium signals were recorded while superfusing the slices with a cocktail of GABAA, GABAB, and adenosine A1 receptor antagonists, i.e., bicuculline methochloride (10 µM), CGP55845 (300 nM), and DPCPX (100 nM), respectively. Fluorometric measurements were analyzed on- and off-line using the Acquis1 computer program (Biologic).
Statistical significance was assessed by paired or unpaired t-test, as appropriate, with P < 0.05 (2-tailed) considered significant. All error values given are mean ± SE.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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In 10- to 12-day-old mice, as shown in the previous study by Maejima et al. (2001), 5-min bath application of the selective mGluR1 and mGluR5 agonist DHPG, at an apparent saturing concentration of 100 µM (Canepari et al. 2004; Pin and Duvoisin 1995; Schoepp et al. 1994), induced a large and significant (P < 0.001) decrease in the mean amplitude of PF-EPSCs to 54.17 ± 5.12% of control (n = 6; Fig. 1, A and B). This suppression was accompanied by a large and significant (P < 0.002) increase in PPF, from 1.35 ± 0.06% in control to 1.86 ± 0.19% at the peak of the DHPG effect. This is in keeping with a presynaptic action of DHPG at PF-PC synapses through retrograde signaling in juvenile mice (Maejima et al. 2001). PF-EPSCs recovered their initial amplitude within <2 min of DHPG application and thereafter were potentiated for several minutes (123.48 ± 8.64% of control on average; Fig. 1, A and B). Very similar results were obtained in 10- to 12-day-old rats because bath application of 100 µM DHPG also induced a reversible decrease in the mean amplitude of PF-EPSCs to 68.22 ± 4.37% of control that was also followed by a transient potentiation of PF-EPSCs (n = 6; Fig. 1C). However, this potentiation was shorter than in 10- to 12-day-old mice and was later followed by a transient, albeit not significant, depression of PF-EPSCs that amounted to 8.98 ± 8.95% on average (Fig. 1C). In both mice and rats, the transient potentiation that followed the initial suppression of PF-EPSCs did not give rise to any significant variation in PPF (data not shown). It was therefore reminiscent of transient potentiations observed in older animals after blockade of endocannabinoid dependent- and endocannabinoid-independent components of DHPG-induced suppression of PF-EPSCs. Accordingly, these potentiating effects are likely to be induced at a postsynaptic level in immature and in nearly mature PCs (see DISCUSSION).
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, DHPG-induced suppression of PF-EPSCs was nearly totally abolished by 30-min bath application of the CB1 receptor antagonist AM-251 (n = 9; Fig. 1B) applied at a saturating concentration of 2 µM (Gatley et al. 1996). In 10- to 12-day-old rats, DHPG-induced suppression of PF-EPSCs was similarly abolished (n = 6; Fig. 1C) in the presence of the CB1 receptor antagonist SR141716-A (Rinaldi-Carmona et al. 1994) at a saturating concentration of 1 µM (Petitet et al. 1996). In both cases, blockade of agonist-induced suppression of PF-EPSCs by either AM-251 or SR141716-A revealed that the transient potentiation induced by DHPG had an earlier onset than that seen in the absence of CB1 receptor antagonists (Fig. 1, B and C). In 10- to 12-day-old rats, the amplitude and time-course of this potentiation were also slightly, although not significantly, increased in the presence of SR141716-A (Fig. 1C). Altogether, these results confirm that the suppression of PF-EPSCs by DHPG in juvenile rodents depends entirely on retrograde release of endocannabinoids (Maejima et al. 2001).
In nearly mature (18–22 days old) rats (n = 10) and for all cells tested, 5-min bath application of 100 µM DHPG induced a transient and significant (P < 0.001) decrease in the amplitude of PF-EPSCs that amounted to 43.23 ± 5.08% of control (Fig. 2, A and B1). Most importantly, this transient suppression of PF-EPSCs was always accompanied by a significant increase in PPF (P < 0.001) from 1.32 ± 0.08 in control conditions to 1.95 ± 0.22 at the peak of the DHPG effect (Fig. 2B2). This is in keeping with a presynaptic site of action of DHPG at PF-PC synapses through retrograde signaling in nearly mature rats (Levenes et al. 2001). However, in this earlier study, suppression of PF-EPSCs of similar amplitude as those reported here were obtained with (S)-DHPG concentrations of only 50 µM (compare Fig. 2B1 of this study with Fig. 2B in Levenes et al. 2001). In pilot experiments, suppression of PF-EPSCs induced by 50 µM (S)-DHPG applications was on average 1.5 times smaller than that achieved with the same concentration in this earlier study (n = 5; Supplementary Fig. S1).1 With 25 µM DHPG, the late (endocannabinoid-independent) component of suppression of PF-EPSCs was nearly totally absent and the initial component was still further reduced in amplitude (n = 5; Supplementary Fig. S1). This suggests that, taking into account the present recording chamber's exchange time, this nominal concentration was too low to fully saturate mGluR1 receptors during 5-min bath applications. Therefore concentrations of 100 µM DHPG that gave rise to more robust suppressions of PF-EPSCs were used throughout this study in nearly mature animals. This was also the case in experiments on juvenile rats and mice to compare results with mature ones in the same experimental conditions.
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30 min with 1 µM SR141716-A only partly inhibited the DHPG-induced suppression of PF-EPSCs. Indeed, mean amplitude decrease was only 22.11 ± 4.30% (n = 14), a value significantly smaller (P < 0.01) than that observed in control conditions (Fig. 2B1). The partial inhibitory effect of 1 µM SR141716-A on the DHPG-induced suppression of PF-EPSCs was unlikely to result from an incomplete blockade of presynaptic CB1 receptors by SR141716-A because bath application of AM-251 at a saturating concentration of 2 µM did not further antagonize this inhibition of PF-EPSCs. Indeed, in the presence of 2 µM AM-251, the mean decrease in PF-EPSC amplitude in the presence of 100 µM DHPG was 26 ± 3.75% (n = 6; data not shown), a value very similar to that obtained in the presence of 1 µM SR141716-A. Moreover and in agreement with our previous study (Levenes et al. 1998), these concentrations of SR141716-A and of AM-251 fully antagonized the depressant effect of 1 µM bath application of the selective CB1 receptor agonist WIN55,212–2 (Devane et al. 1988) on PF-EPSCs (n = 3 in each case; data not shown).
Finally, and most importantly, in the presence of CB1 receptor antagonists, the remaining suppression of PF-EPSCs was still accompanied by a significant (P < 0.01) increase in PPF that amounted to 17%, i.e., from 1.29 ± 0.05 in control conditions to 1.51 ± 0.08 at the peak of the DHPG effect (Fig. 2B2). These latter results suggest that suppression of PF-EPSCs by DHPG in nearly mature rats not only involves activation of presynaptic CB1 receptors like in juvenile animals (Maejima et al. 2001), but also involves at least one other presynaptic mechanism. In contrast, kinetics of PF-EPSCs were not significantly affected. Thus mean values of the 10–90% rise time were 2.06 ± 0.21 and 1.90 ± 0.20 ms in control conditions and at the peak of DHPG-induced suppression of PF-EPSCs, respectively, and mean time constants of decay had values of 11.39 ± 0.75 and 10.90 ± 0.79 ms in the same conditions. Although kinetics are certainly severely biased in nearly mature PCs by dendritic filtering of synaptic currents, these data do not suggest that postsynaptic AMPA receptors at PF-PC synapses are sizably affected during the endocannabinoid-independent component of DHPG-induced suppression of PF-EPSCs (see DISCUSSION).
Sensitivity of the CB1 receptor–independent component of agonist-induced suppression of PF-EPSCs to a Glu uptake blocker and to a desensitizing KA receptor agonist in nearly mature rats
In nearly mature rats, retrograde release of Glu by PCs participates in DSE at PF-PC synapses through activation of presynaptic KA receptors that include GluR6 receptor subunits (Crepel 2007). In keeping with this recent finding, the previous study by Levenes et al. (2001) already suggested that activation of postsynaptic mGluR1 decreases PF-EPSCs through retrograde release of Glu and activation of presynaptic ionotropic Glu receptors borne by PFs. Accordingly, in 18- to 22-day-old rats, the endocannabinoid-independent component of agonist-induced suppression of PF-EPSCs should be enhanced in the presence of the Glu uptake inhibitor D-TBOA and suppressed by pharmacological blockade of presynaptic KA receptors.
In the presence of 1 µM SR141716-A and as expected for a Glu uptake inhibitor, application of 100 µM D-TBOA increased the amplitude of PF-EPSCs (Fig. 3A1). This increase ranged between 8 and 76% depending on cells, with a significant mean increase of 34.90 ± 9.79% (n = 9; P < 0.01). As seen in a previous study (Crepel 2007), the rather large variability of potentiating effects of D-TBOA on PF-EPSCs might result from the patterned expression of PC Glu transporters EAAT4 in rat cerebellar cortex (Wadiche and Jahr 2005). In all cells, this effect was accompanied by marked changes in EPSC kinetics (Fig. 3A1), probably because of slower clearance of Glu from synaptic cleft. At the plateau of the D-TBOA effect, application of 100 µM DHPG decreased the mean amplitude of PF-EPSCs that amounted to 39.78 ± 4.63% of control amplitude before DHPG application (Fig. 3, A2 and B1). This decrease in amplitude was significantly larger (P < 0.01) than that observed in the presence of SR141716-A alone (Fig. 3B1) and was accompanied by a significantly larger increase in PPF (P < 0.02) because PPF increased from 1.24 ± 0.17 in SR141716-A + D-TBOA containing medium to 1.77 ± 0.15 at the peak of the DHPG effect (Fig. 3B2). Although slower Glu clearance in the presence of D-TBOA may complicate the interpretation of this difference, the fact that D-TBOA alone did not induce any significant change in PPF (Crepel 2007) suggests that D-TBOA did not affect the presynaptic machinery to such an extent as to sizably affect the level of suppression of PF-EPSCs by DHPG. Therefore these results are consistent with the assumption that retrograde release of Glu participates, together with retrograde release of endocannabinoids, to the depressant effect of DHPG on PF-EPSCs in nearly mature rats. However, it should be noted that group 1 mGluRs are not restricted to PCs but are also found, for instance, on glial cells (Angulo et al. 2004; Karakossian and Otis 2004; Parpura et al. 1994).
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, involvement of presynaptic KA receptors in agonist-induced suppression of PF-EPSCs in 18- to 22-day-old rats was tested by using SYM 2081, a potent ligand that, at micromolar concentrations, selectively blocks KA-induced currents through a process of agonist-induced desensitization (Cho et al. 2003; Cossart et al. 2002; DeVries 2000; Epsztein et al. 2005; Li et al. 1999; Zhou et al. 1997). In the presence of 1 µM SR141716-A, superfusing the slices with 10 µM SYM 2081 did not lead to large changes in PF to PC synaptic transmission, except for a significant (P < 0.001) increase in basal PPF, which amounted to 1.53 ± 0.07% (n = 11) compared with 1.29 ± 0.05% in control conditions. In marked contrast, SYM 2081 totally inhibited the late phase of the endocannabinoid-independent suppression of PF-EPSCs induced by bath application of 100 µM DHPG, and the same was true for the associated increase in mean normalized PPF (n = 11; Fig. 4, A and B). Nearly identical results were obtained in six other cells with 10 µM SYM 2081 + 100 µM D-APV (data not shown). These results strongly suggest that, like for DSE in nearly mature rats, a late phase of the endocannabinoid-independent suppression of PF-EPSCs induced by bath application of 100 µM DHPG involves activation of presynaptic KA receptors.
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) was further added to the bath (n = 8; Fig. 4, A and B). This suggests that the early component of the endocannabinoid-independent suppression of PF-EPSCs is caused by activation of presynaptic GABAB receptors (Dittman and Regehr 1997) by GABA released by molecular layer inhibitory interneurons, as well as to release of NO from these same cells (Bredt et al. 1990; Shin and Linden 2005; Vincent and Kimura 1992). Indeed, one knows that molecular layer inhibitory interneurons bear group I mGluRs (Baude et al. 1993; Karakossian and Otis 2004) and are therefore likely to be stimulated by DHPG, thus leading to release of GABA and of NO, which in turn transiently depress PF-EPSCs by presynaptic mechanisms (Blond et al. 1997). This presynaptic mGluR1 receptor/NO cascade is reminiscent of the presynaptic N-methyl-D-aspartate (NMDA) receptor/NO cascade found in molecular layer inhibitory interneurons and involved in the induction of cerebellar long-term depression (LTD) through cGMP-dependent inhibition of postsynaptic protein phosphatases (Shin and Linden 2005). Therefore NO released by molecular layer inhibitory interneurons is likely to be involved in both short-term presynaptic and long-term postsynaptic modulation of PF-PC synaptic transmission, depending on additional mechanisms involved, such as activation of postsynaptic mGluR1, protein kinase C
, and phosphorylation of ser-880 on the AMPA receptor subunit GluR2 for cerebellar LTD (references in Shin and Linden 2005). Finally, blockade of the early component of the endocannabinoid-independent suppression of PF-EPSCs in the eight PCs mentioned above unmasked a short-term potentiation of PF-EPSCs in five of them, whereas no such potentiation was seen in the other three. On average, this potentiation amounted to 130.13 ± 10.11% of control and was accompanied by a slight, although nonsignificant, mean normalized PPF decrease (Fig. 4, A and B). Altogether, these results suggest that, like for DSE (Crepel 2007), a late phase of agonist-dependent suppression of PF-EPSCs in nearly mature rats depends on retrograde release of Glu by PCs and activation of presynaptic KA receptors located on PFs. Sensitivity of the CB1 receptor–independent component of suppression of PF-EPSCs by DHPG to nonsaturating concentrations of CNQX and GYKI in nearly mature rats
Because SYM 2081 is a desensitizing KA receptor agonist rather than a genuine KA receptor antagonist, it was important to confirm the involvement of presynaptic KA receptors in the late phase of agonist-induced suppression of PF-EPSCs. We reasoned that the concentration of Glu achieved at the level of presynaptic KA receptors involved in the CB1 receptor–independent component of agonist-induced suppression of PF-EPSCs is likely to be much lower than that seen by postsynaptic AMPA receptors during PF-PC EPSCs. If so, it is possible that a nonsaturating concentration of the competitive AMPA/KA antagonist CNQX (Honoré et al. 1988) that only partly block PF-EPSCs is sufficient to fully antagonize presynaptic KA receptors and thus inhibit agonist-induced suppression of PF-EPSCs.
Indeed, bath application of 1 µM CNQX in the presence 1 µM SR141716-A reduced the amplitude of PF-EPSCs to 36.33 ± 3.76% of the control value (n = 11). Unexpectedly, this effect was accompanied by a large and highly significant (P < 0.001) PPF increase, from 1.30 ± 0.08 to 1.75 ± 0.14 (Fig. 5A1) that was reminiscent of that observed for basal PPF in the presence of SYM 2081 in nearly mature rats and in GluR6–/– mice (Crepel 2007). In keeping with this latter result that suggests a presynaptic site of action of CNQX in addition to its well-established postsynaptic effect, mean CV of PF-EPSCs also significantly (P < 0.001) increased from to 0.045 ± 0.006 in control to 0.081 ± 0.012 at the steady state of the depressant effect of CNQX (n = 9; Fig. 5B).
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To preclude that the strong reduction in PF-EPSC amplitude by CNQX was not solely responsible for the lack of suppression of PF-EPSCs by DHPG in the experiments reported above, we also studied the effect of nonsaturating concentrations of GYKI, a noncompetitive and more selective AMPA receptor antagonist (Bureau et al. 1999; Renard et al. 1995; Wilding and Huettner 1995). On average, bath application of 20 µM GYKI reduced the amplitude of PF-EPSCs to 42.63 ± 3.92% of control values (n = 11), a decrease nonsignificantly different from that obtained with 1 µM CNQX. Like for experiments with CNQX, this decrease in EPSC amplitude was also accompanied by a significant (P < 0.01) PPF increase, although about only one half of that obtained in the presence of CNQX (Fig. 5A1). However, and in marked contrast with results obtained with CNQX, mean CV of PF-EPSCs did not significantly increase during the depressant effect of GYKI on PF-EPSCs, because mean CV values during the control period and at the steady state of the effect of GYKI were 0.046 ± 0.005 and 0.051 ± 0.006, respectively (Fig. 5B).
In 8 of the 11 tested cells, GYKI did not significantly inhibit the CB1 receptor–independent suppression of PF-EPSCs induced by bath application of 100 µM DHPG, because the mean decrease in peak amplitude of PF-EPSCs was 28.48 ± 7.07% compared with 22.11 ± 4.30% (n = 14) in the presence of SR141716-A alone (Fig. 5A2). Moreover, the time-course of PF-EPSC suppression was unchanged (cf. Fig. 2B1 and 5A2). In the remaining three cells, GYKI totally abolished the CB1 receptor–independent suppression of PF-EPSCs induced by bath application of 100 µM DHPG, which unmasked a short-term potentiation of PF-EPSCs (Fig. 5A2). Because there was no apparent difference between these two groups of cells with respect to the effect of GYKI on PF-EPSC amplitude and on basal PPF, DHPG results were pooled, leading to a mean suppressing effect of this compound on peak amplitude of PF-EPSCs that amounted to 20.83 ± 5.76%. This value was very close to that obtained in the presence of SR141716-A alone. Accordingly, DHPG suppression of PF-EPSCs was still accompanied by a significant (P < 0.05) and near 20% increase in mean PPF, from 1.54 ± 0.08 in the presence of SR141716-A + GYKI to 1.82 ± 0.18 at the peak of DHPG effect (Fig. 5A1).
Taken together, CNQX and GYKI results fully confirm that presynaptic KA receptors are likely to be involved in CB1 receptor–independent suppression of PF-EPSCs by mGluR1 agonists (see DISCUSSION).
Finally, because PF-EPSC amplitude did not reach a steady state during DHPG application (Figs. 2B1 and 5A2), no attempt was made to apply the CV method here. In contrast, such a near steady state was achieved during the endocannabinoid-independent component of DSE (Crepel 2007) and was accompanied by a significant increase in CV (see Supplementary Results and Supplementary Fig. S2), thus confirming involvement of presynaptic mechanisms in the CB1 receptor–independent component of DSE (Crepel 2007).
DHPG sensitivity of PF-EPSCs in wild-type and GluR6–/– mice
Presynaptic KA receptors located on PFs are likely to be heteromeric constructions that include GluR6 and KA2 receptor subunits (Lerma et al. 2001; Petralia et al. 1994). Their activation up- or down-regulates Glu release depending on agonist concentration (Delaney and Jahr 2002). Agonist-induced suppression of PF-EPSCs was studied in nearly mature GluR6–/– mice and compared with that of wild-type mice of the same strain (see METHODS), with the assumption that invalidating GluR6 receptor subunits renders presynaptic KA receptors nonfunctional (Ruiz et al. 2005). All experiments in GluR6–/– mice were performed in the presence of 50 µM D-APV to minimize possible developmental compensations by presynaptic NMDA receptors and in the presence of 1 µM SR141716-A to focus on the endocannabinoid-independent component of agonist-induced suppression of PF-EPSCs. For comparison, all experiments in wild-type mice were also performed in the presence of 50 µM D-APV and of 1 µM SR141716-A.
In 22- to 24-day-old wild-type mice, 5-min bath application of 100 µM DHPG induced an endocannabinoid-independent suppression of PF-EPSCs of similar amplitude and duration as in nearly mature rats, with a mean peak amplitude decrease of 17.62 ± 2.76% (n = 6; Fig. 6A). This suppression was accompanied by a significant (P < 0.001) increase in mean normalized PPF, from 100.39 ± 3.66% in control conditions to 116.27 ± 3.24% at the peak of the DHPG effect (Fig. 6B).
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), no major alteration in PF to PC synaptic transmission was seen, except for basal PPF, which amounted to 1.54 ± 0.01% (n = 6) compared with 1.31 ± 0.05% in control mice, the difference being highly significant (P < 0.001). In contrast, the late phase of the endocannabinoid-independent suppression of PF-EPSCs by DHPG and of its associated increase in PPF was strongly inhibited in all cells, whereas the initial phase remained largely unaffected (Fig. 6, A and B). Therefore these results are very similar to those obtained in experiments performed in the presence of SYM 2081 and of CNQX and fully confirm that the late phase of the endocannabinoid-independent suppression of PF-EPSCs by DHPG depends on retrograde release of Glu. Moreover, they suggest that Glu acts through activation of KA receptors that include GluR6 subunits.
GDP-S sensitivity of the CB1 receptor–independent component of agonist-induced suppression of PF-EPSCs in nearly mature rats
As mentioned before, group 1 mGluRs are not restricted to PCs but are also found on various cell types including granule cells, molecular layer interneurons, and glial cells (Angulo et al. 2004; Baude et al. 1993; Parpura et al. 1994). As such, glutamate release from these cells might participate in DHPG-induced suppression of PF-EPSCs. In an attempt to exclude this possibility, we selectively blocked G protein activity in the postsynaptic compartment by dialyzing PCs through the patch pipette for 30 min after break-in with a conventional K-gluconate internal solution with 4 mM GDP-S added (Galante and Diana 2004); GDP-S is a nonhydrolyzable GTP analog that inhibits G protein activity. Experiments were performed in the presence of 1 µM SR141716-A to focus on the endocannabinoid-independent suppression of PF-EPSCs. In five of the six cells tested, the late phase of the endocannabinoid-independent suppression of PF-EPSCs induced by bath application of 100 µM DHPG was abolished and replaced by a short-term potentiation of PF-EPSCs, whereas its initial phase was much less affected (Fig. 7A). In these five cells, the late phase of mean normalized PPF increase associated with the endocannabinoid-independent suppression of PF-EPSCs was also inhibited and replaced by a nonsignificant 7.72 ± 2.83% decrease of the mean normalized PPF (Fig. 7B). In the remaining PC, the late phase of the endocannabinoid-independent suppression of PF-EPSCs was only partly abolished (data not shown). In contrast, in five other PCs dialyzed during the same period of time after break-in with a conventional K-gluconate internal solution without GDP-S, a clear-cut endocannabinoid-independent suppression of PF-EPSCs and associated PPF increase were still induced by bath application of 100 µM DHPG (Fig. 7, A and B).
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Ryanodine sensitivity of the CB1 receptor–independent component of agonist-induced suppression of PF-EPSCs and of DSE in nearly mature rats
Depolarization-induced potentiation of inhibition (DPI) also operates through Glu release from depolarized PCs. In this case, Glu activates presynaptic NMDA receptors, resulting in a slow build-up and decay (over several minutes) of calcium release from presynaptic ryanodine-sensitive calcium stores (Duguid and Smart 2004). As for DPI, the KA-dependent components of agonist-induced suppression of PF-EPSCs and of DSE at PF-PC synapses might therefore involve such a mechanism in nearly mature rats, as suggested previously (Crepel 2007; but see also Carter et al. 2002). This hypothesis was tested by the following experiments.
In the presence of 1 µM SR141716-A and of 100 µM ryanodine, the CB1 receptor–independent suppression of PF-EPSCs by bath application of 100 µM DHPG was strongly inhibited and replaced by a transient potentiation of PF-EPSCs, 112.84 ± 9.43% on average (n = 6; Fig. 8A1). In these cells, PPF increase associated with this CB1 receptor–independent suppression of PF-EPSCs was also strongly inhibited (Fig. 8A2). Interestingly, ryanodine also markedly reduced basal PPF because its mean value was only 1.09 ± 0.06 (n = 6; Fig. 8A2), thus suggesting that presynaptic ryanodine-sensitive calcium stores also contribute to basal PPF at PF-PC synapses in nearly mature rats.
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; Fig. 8, B1 and B2). Here again, bath application of 100 µM ryanodine also markedly reduced basal PPF because the mean paired-pulse ratio was only 0.90 ± 0.04 in these experiments (n = 6). The fact that the early component of DSE that was partly resistant to bath application of ryanodine was not accompanied by significant changes in paired-pulse ratio (Fig. 8, B1 and B2) suggests in turn that it might be partly postsynaptic in origin, i.e., caused by a transient ionic unbalance after large depolarizing steps in postsynaptic cells (Crepel 2007). Altogether, the marked effects of ryanodine on agonist-induced suppression of PF-EPSCs and on DSE at PF-PC synapses suggests that, as for DPI, their prolonged duration involves long-lasting calcium release from presynaptic ryanodine-sensitive calcium stores, after an initial and more short-lived activation of presynaptic KA receptors. Moreover, the fact that ryanodine also partly inhibited the early phase of the CB1 receptor–independent suppression of PF-EPSCs by DHPG, i.e., the portion of the response that was resistant to bath application of SYM 2081 (cf. Figs. 4A and 8B1), suggests in turn that ryanodine also inhibits the presynaptic GABAB- and NO-dependent components of agonist-induced suppression of PF-EPSCs.
In these experiments, inhibition of the Glu-dependent component of the agonist-induced suppression of PF-EPSCs by ryanodine might also result from an inhibition of calcium release from postsynaptic ryanodine-sensitive calcium stores (Carter et al. 2002; Isokawa and Alger 2006) that would in turn inhibit retrograde release of Glu from PCs. To compensate, at least partly, for these postsynaptic effects, we used 2-s rather than 1-s depolarizing pulses from –70 to 0 mV in another series of DSE experiments in the presence of ryanodine. Indeed, this duration was likely to give rise to postsynaptic calcium transients of similar amplitude to those observed with 1-s depolarizing pulses in the absence of ryanodine (Fig. 9B1). As shown in Fig. 8B1, the Glu-dependent component of DSE (Crepel 2007) was still significantly inhibited (n = 9; P < 0.01), whereas the early residual DSE was not significantly different from that induced in the same conditions with 1-s depolarizing pulses (Fig. 8B1).
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) on the other hand. The latter protocol was directly derived from that used by Duguid and Smart (2004) to show involvement of presynaptic calcium-induced calcium release in DPI. Sensitivity to ryanodine of pre- and postsynaptic calcium signaling involved in the CB1 receptor–independent component of DSE in nearly mature rats
PFs loaded with Fluo-4FF-AM were subjected to focal superperfusion of 20 µM domoate for 1 min. This elicited a transient increase in the cytosolic calcium signal in these fibers that peaked shortly after the end of domoate application and relaxed slowly thereafter in <30 min (n = 6; Fig. 9, A1 and A2). Ryanodine (100 µM) present in the bath for 30 min markedly reduced the duration of domoate-induced calcium transients, whereas the peak calcium transient was only marginally affected (n = 5; Fig. 9, A1 and A2). Indeed, peak F/F in control conditions and in the presence of ryanodine were 3.58 ± 0.10 and 3.42 ± 0.94%, respectively; these values are not significantly different. To further quantify differences in calcium transients recorded in control conditions and in the presence of ryanodine, we determined for each cell 
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(F/F for each point of F/F plots) between the beginning of domoate application and 30 min thereafter. We averaged these values for all cells recorded in a given condition (see Crepel 2007). Mean was significantly lower in the presence of ryanodine, because it amounted to only 0.61 ± 0.14 compared with 1.52 ± 0.30 in its absence (P < 0.05). These results strongly suggest that prolonged calcium release from presynaptic ryanodine-sensitive stores occurs in PFs after brief activation of presynaptic KA receptors.
In control bathing medium, calcium signals induced in PC dendrites by the above described DSE protocol (depolarizing voltage steps from –70 to 0 mV for 1 s) peaked to F/F = 14.10 ± 2.85% at the end of the depolarizing steps (Fig. 9B1) and rapidly decayed over the course of 10–15 s (n = 6; Fig. 9B2). Superfusion of the slices for 30 min with 100 µM ryanodine significantly (P < 0.05) inhibited these depolarization-induced calcium transients because mean F/F was now only 7.77 ± 1.63% (n = 6; Fig. 9, B1 and B2). Differences between control and ryanodine results were further quantified by determining again, for each cell, mean values calculated from the start of the depolarizing step until 4 s after, i.e., the period of time during which F/F plots in control conditions and in the presence of ryanodine appeared clearly different (Fig. 9B2). The mean value was significantly lower (P < 0.05) with ryanodine (4.56 ± 1.17) than in its absence (8.52 ± 1.27). Here again and in keeping with previous results by Carter et al. (2002), this suggests that calcium release from intracellular ryanodine-sensitive stores participates in calcium transients induced in PC dendrites by short depolarizing voltage steps identical as those used to induce DSE (Crepel 2007). However, these calcium transients were much shorter than those induced in PFs by domoate application, and moreover, they were also much shorter than the duration of the KA-sensitive component of DSE in nearly mature rats (Crepel 2007). Therefore electrophysiological and fluorometric experiments with ryanodine clearly suggest that prolonged calcium release from presynaptic ryanodine-sensitive stores is responsible for the prolonged duration of DSE and of agonist-induced suppression of PF-EPSCs in nearly mature rats.
Origin of the lack of the Glu-dependent component of agonist-induced suppression of PF-EPSCs in immature rats
Depolarization-induced potentiation of inhibition that operates through Glu release from depolarized PCs is strictly dependent on the activity of surrounding Glu transporters (Duguid and Smart 2004). Therefore the lack of a Glu-dependent component of agonist-induced suppression of PF-EPSCs in juvenile animals might simply result from an unbalance between Glu release and Glu uptake in favor of the latter at early developmental stages. In 10- to 12-day-old rats and in the presence of 1 µM SR141716-A, bath application of the Glu uptake inhibitor D-TBOA (100 µM) did not unmask any sizeable Glu-dependent component of agonist-induced depression of PF-EPSCs (n = 5; Fig. 10A). However, the transient potentiation elicited by DHPG was significantly (P < 0.001) inhibited compared with that observed in the presence of SR141716-A alone (Fig. 10A), suggesting that it was counterbalanced by a DHPG-induced suppression of PF-EPSCs of similar amplitude and time-course. Thus and as shown for DSE (Crepel 2007), the absence of a Glu-dependent component of agonist-induced depression of PF-EPSCs in juvenile rats may be partly explained by the activity of surrounding Glu transporters. However, this absence might also be caused by other factors such as incomplete maturation of presynaptic KA receptors or of presynaptic calcium-induced calcium release, because D-TBOA failed to reveal any fully developed KA-dependent component of agonist-induced suppression of PF-EPSCs in these animals. Therefore fluorometric experiments were also performed to test these hypotheses.
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F/F was only 1.82 ± 0.21%. Moreover, calcium transients relaxed within <2 min instead of within 30 min in nearly mature rats (cf. Figs. 9A2 and 10B1). Therefore these results are in agreement with the lack of a Glu-dependent component of agonist-induced suppression of PF-EPSCs in immature rats and suggest that presynaptic KA receptors and/or calcium release from internal stores are not yet fully developed at this early developmental stage.
In contrast, when domoate application was performed in the presence of 100 µM D-TBOA, cytosolic calcium signals were significantly (P < 0.001) larger (F/F = 4.74 ± 0.53%) and now decayed over 15 min (n = 5; Fig. 10B1). Moreover, this decay was markedly shortened when 100 µM ryanodine was also present in the bath (n = 5; Fig. 10B1), so that the mean value was significantly lower (P < 0.05) in the presence of ryanodine (0.96 ± 0.30) than in its absence (2.65 ± 0.47). These results therefore suggest that at least some calcium-induced calcium release may be triggered in immature PFs.
However, the prominent effect of D-TBOA on cytosolic calcium signals was puzzling. Indeed, and to the best of our knowledge, significant uptake of this agonist is unlikely. However, domoate is known to induce release of excitatory amino acids from cultured cerebellar granule cells through reversal of the Glu transporter (Berman and Murray 1997). Therefore the possibility exists that, in these experiments, domoate not only acts through activation of presynaptic KA receptors, but also activates other presynaptic Glu receptors after release of Glu from the superfused molecular layer. We therefore tested this possibility by studying the effect of bath-applied D-APV (50 µM) on calcium transients elicited in PF by focal superfusion of the slices for 1 min with 20 µM domoate in the presence of D-TBOA. We performed these experiments because NMDA receptors are characterized by their high affinity for Glu and are probably present and functional on PFs (Casado et al. 2000). However, this conclusion has been challenged recently (Shin and Linden 2005) and, to date, it still has not been established by electron microscopic analysis that NMDA receptors exist on PFs. As shown in Fig. 10B2, D-APV shortened calcium transients induced by domoate in the presence of D-TBOA (n = 6) to nearly the same extent as that observed with ryanodine because mean values were not significantly different, i.e., 0.96 ± 0.18 and 0.96 ± 0.30, respectively. This latter result indicates that activation of presynaptic NMDA receptors probably contributes to calcium transients induced by domoate in the presence of Glu uptake blockers, thus corroborating the aforementioned hypothesis, and also supports the previous finding that NMDA receptors are present on PFs (Casado et al. 2000). Furthermore, these results also strongly suggest that activation of NMDA receptors is necessary to trigger calcium-induced calcium release in immature PFs. In contrast, D-APV did not significantly affect calcium transients induced by domoate in control bathing medium in 20- to 22-day-old rats (n = 4; data not shown), suggesting that presynaptic NMDA receptors are not recruited in this case.
In 12-day-old rats, the mean values of calcium transients elicited in the presence of D-TBOA and D-APV or in the presence of D-TBOA and ryanodine were still significantly (P < 0.05) higher (0.96 ± 0.18 and 0.96 ± 0.30, respectively) than in control medium (0.10 ± 0.02; Fig. 10B2). It is therefore likely that an additional mechanism participates to calcium transients induced by domoate in PFs in the presence of D-TBOA. One hypothesis is that Glu uptake blockers lead to a progressive accumulation of Glu within slices that tonically activates granule cells. Such activity might therefore increase basal calcium concentration within PFs and facilitate induction of calcium-induced calcium release on activation of presynaptic KA receptors. If this were so, prolonged calcium transients induced by domoate in the presence of D-TBOA and D-APV should be no longer observed in the presence of TTX. However, in six experiments performed in the presence of 1 µM TTX, calcium transients induced by focal application of 20 µM domoate in the presence of both 100 µM D-TBOA and 50 µM D-APV was not significantly altered compared with that induced when TTX was omitted (Fig. 10B3). At the moment, we have no other plausible explanation to explain that calcium transients elicited in the presence of D-TBOA and D-APV or i
