Miniature Synaptic Events Elicited by Presynaptic Ca²+ Rise Are Selectively Suppressed by Cannabinoid Receptor Activation in Cerebellar Purkinje Cells

Cannabinoid agonist suppresses mIPSC frequency but not mEPSC frequency with 2 mm [Ca²⁺]_o

Previous reports have shown that mIPSCs recorded in PCs are consistently suppressed by cannabinoid agonists at a normal [Ca²⁺]_o (2 mm) (Takahashi and Linden, 2000; Diana et al., 2002). Moreover, the mIPSCs of a PC are suppressed by strong depolarization of the cell itself that triggers endocannabinoid-mediated retrograde suppression of inhibitory synaptic inputs known as depolarization-induced suppression of inhibition (DSI) (Llano et al., 1991; Vincent and Marty, 1993; Diana et al., 2002; Yoshida et al., 2002; Diana and Marty, 2003). In marked contrast, the mEPSCs of PCs are not affected by cannabinoid agonists at a normal [Ca²⁺]_o (Takahashi and Linden, 2000) (but see Levenes et al., 1998). We began by analyzing the effects of WIN55,212-2 (5 μm), a cannabinoid agonist, on mEPSCs and mIPSCs recorded in the presence of TTX (1 μm). With 2 mm Ca²⁺_o, WIN55,212-2 had no effect on the frequency and amplitude of mEPSCs (Fig. 1A,C,E). The mean control frequency and amplitude were 0.53 ± 0.14 Hz and 17.6 ± 1.0 pA, respectively (n = 5). WIN55,212-2 affected neither the frequency (89.1 ± 15.0% of the control) nor the amplitude (101.6 ± 1.5% of the control) of mEPSCs (n = 5; p = 0.24 and 0.35, respectively). In marked contrast, WIN55,212-2 reduced the mean mIPSC frequency (Fig. 1B,D,F). The mean baseline frequency was 4.5 ± 0.8 Hz (n = 9). The mIPSC frequency with WIN55,212-2 was 56.9 ± 5.2% of the control (n = 9; p < 0.01). This suppression was reversed to 119.7 ± 8.5% of the control (n = 9) by subsequent application of SR141716A (2 μm), a selective cannabinoid antagonist (Fig. 1D). Conversely, mean mIPSC amplitude did not change significantly (Fig. 1F). The mean baseline amplitude was 53.1 ± 9.5 pA and that with WIN55,212-2 was 101.4 ± 3.0% of the control (n = 9; p = 0.88). These results are consistent with a previous report that the mIPSCs of PCs are sensitive to cannabinoids at a normal [Ca²⁺]_o but the mEPSCs are not (Takahashi and Linden, 2000).

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Figure 1.

Effects of WIN55,212-2, a cannabinoid agonist, and SR141716A, a cannabinoid antagonist, on mEPSCs and mIPSCs at normal [Ca²⁺]_o. A, C, E, Representative data showing the effect of WIN55,212-2 on mEPSCs with 2 mm Ca²⁺_o. A, Sample traces taken from the indicated period in C and E from the same PC. C, Average time plot of normalized mEPSC frequency (n = 5). WIN55,212-2 (5μm) and SR141716A (2μm) had no effect on mEPSC frequency. E, Average time plot of normalized mEPSC amplitude (n = 5). WIN55, 212-2 (5 μm) and SR141716A (2 μm) had no effect on mEPSC amplitude. B, D, F, Representative data showing the effect of WIN55,212-2 on mIPSCs with 2 mm Ca²⁺_o. B, Sample traces were taken from periods labeled in D and F. D, Average time plot of normalized mIPSC frequency (n = 4). WIN55,212-2 (5 μm) reduced mIPSC frequency, and SR141716A (2μm) reversed the reduction. F, Average time plot of normalized mIPSC amplitude (n = 4). WIN55,212-2 (5 μm) and SR141716A (2 μm) had no effect on mIPSC amplitude. Error bars represent SEM.

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Figure 2.

Ca²⁺_o dependency of mEPSC and mIPSC. A, C, E, Effect of changing [Ca²⁺]_o on mEPSC frequency. A, Sample mEPSCs recorded with 2 mm Ca²⁺_o and 0 mm Ca²⁺_o. C, Sample mEPSCs recorded with 2 mm Ca²⁺_o and 5 mm Ca²⁺_o. E, Summary bar graph for the effects of changing [Ca²⁺]_o on mEPSC frequency. Data with 0 mm Ca²⁺_o (n = 12), with 5 mm Ca²⁺_o (n = 52), and with 5 mm Ca²⁺_o solution preincubated with EGTA-AM (100 μm) (n = 11) are normalized to those with 2 mm Ca²⁺_o. B, D, F, Effect of changing [Ca²⁺]_o on mIPSC frequency. B, Sample mIPSCs recorded with 2 mm Ca²⁺_o and with 0 mm Ca²⁺_o EGTA-containing solution preincubated with BAPTA-AM. D, Sample mIPSCs recorded with 2 mm Ca²⁺_o and with 5 mm Ca²⁺_o. F, Summary bar graph for the effect of changing [Ca²⁺]_o on the mIPSC frequency. Data with EGTA-containing 0 mm Ca²⁺_o solution preincubated with BAPTA-AM (n = 6), with EGTA-containing 0 mm Ca²⁺_o solution (n = 6), and with 5 mm Ca²⁺_o (n=7) are normalized to those with 2 mm Ca²⁺_o. *p < 0.05, **p < 0.01, t test. Error bars represent SEM.

Effect of changing [Ca²⁺]_o on mEPSC and mIPSC

To elucidate the cause of the apparent difference in the cannabinoid sensitivity between mEPSC and mIPSC, we recorded miniature responses at various [Ca²⁺]_o. The mean frequency of mIPSC was decreased as the [Ca²⁺]_o was lowered from 2 to 0 mm (Fig. 2B,F). This is consistent with the previous report by Llano et al. (2000) that mIPSCs recorded in PCs with 2 mm Ca²⁺_o include those elicited by spontaneous Ca²⁺ release from intracellular Ca²⁺ stores (Llano et al., 2000). These Ca²⁺-enhanced mIPSCs seemed to remain even after the slices were perfused with Ca²⁺-free solution containing 200 μm EGTA for >30 min, because the mIPSC frequency was further suppressed when application of EGTA-containing, Ca²⁺-free solution was combined with presynaptic infusion of BAPTA-AM (100 μm; see Materials and Methods) (Fig. 2F). Incubation with BAPTA-AM in 2 mm Ca²⁺_o solution reduced mIPSC frequency (normalized mIPSC frequency was 67.2 ± 8.5%; n = 6; p < 0.01). However, subsequent perfusion with EGTA-containing, Ca²⁺-free solution further reduced mIPSC frequency to 16.4 ± 2.3% of the control, and this was 28.0 ± 4.2% of the value with BAPTA-AM in 2 mm Ca²⁺_o solution (n = 6; p < 0.01). As reported previously, mIPSC with 2 mm Ca²⁺_o was not affected by Cd²⁺ (100 μm), a nonselective blocker of voltage-dependent Ca²⁺ channels (VDCCs) (Llano et al., 2000; Harvey and Stephens, 2004), indicating that VDCCs were not involved in such spontaneous Ca²⁺ rise (the mean frequency and amplitude were 4.0 ± 0.6 Hz and 61.4 ± 10.6 pA with 100 μm Cd²⁺ compared with 3.9 ± 0.7 Hz and 61.2 ± 10.2 pA in control; n = 5; p = 0.87 and 0.98, respectively). In marked contrast, mean mEPSC frequency did not change after the [Ca²⁺]_o was switched from 2 to 0 mm (Fig. 2A,E). Consistent with a previous report (Chen and Regehr, 1997), mEPSC was also not affected by Cd²⁺ (100 μm) (the mean baseline frequency and amplitude was 0.78 ± 0.35 Hz and 17.2 ± 0.7 pA compared with 0.83 ± 0.24 Hz and 17.6 ± 0.7 pA in control; n = 4; p = 0.84 and 0.87, respectively). These results suggest that, with 2 mm Ca²⁺_o, mEPSCs mainly consist of miniature events independent of spontaneous or persistent presynaptic [Ca²⁺]_i rise (we hereafter term such mEPSCs “basal mEPSCs”) and do not include miniature events elicited by presynaptic [Ca²⁺]_i rise (we hereafter term such mEPSCs “Ca²⁺-enhanced mEPSCs”). In contrast, mIPSCs consist of both “basal mIPSCs” and “Ca²⁺-enhanced mIPSCs” with 2 mm Ca²⁺_o.

If cannabinoid agonists preferentially suppress Ca²⁺-enhanced miniature events, this could account for the differential sensitivities of mEPSC and mIPSC to cannabinoid agonists with 2 mm Ca²⁺_o. In the subsequent sections, we tested this hypothesis by addressing the following two questions. First, are Ca²⁺-enhanced mEPSCs suppressed by cannabinoid agonists? Second, even after Ca²⁺-enhanced mIPSCs are eliminated, are the remaining mIPSCs (i.e., basal mIPSCs) suppressed by cannabinoid agonists?

Emergence of Ca²⁺-enhanced mEPSC by raising [Ca²⁺]_i

As reported by Llano et al. (2000), changing the [Ca²⁺]_o from 2 to 5 mm resulted in a marked increase in the frequency (15.4 ± 2.7 Hz with 5 mm Ca²⁺_o compared with 4.9 ± 0.7 Hz with 2 mm Ca²⁺_o; n = 7; p < 0.01) (Fig. 2D,F) but not in the amplitude of Ca²⁺-enhanced mIPSCs (mean amplitude, 60.2 ± 8.4 pA with 5 mm Ca²⁺_o compared with 53.5 ± 6.5 pA with 2 mm Ca²⁺_o; n = 7; p = 0.15). We first examined whether Ca²⁺-enhanced mEPSC could be also elicited by raising the [Ca²⁺]_o. As expected, mEPSC frequency markedly increased after changing the [Ca²⁺]_o from 2 to 5 mm (0.59 ± 0.14 Hz with 2 mm Ca²⁺_o and 6.2 ± 0.5 Hz with 5 mm Ca²⁺_o; n = 52; p < 0.01) (Fig. 2C,E) and rapidly recovered to the initial value during reperfusion with 2 mm Ca²⁺_o solution. There was no significant change in the mean amplitude (17.5 ± 0.4 pA with 2 mm Ca²⁺_o and 17.9 ± 0.4 pA with 5 mm Ca²⁺_o; n = 52; p = 0.85). The increase in mEPSC frequency was blocked by preincubation of the slices with EGTA-AM (100 μm) (1.0 ± 0.2 Hz; n = 11; see Materials and Methods) (Fig. 2E), indicating that it required Ca²⁺ rise in the presynaptic terminals. The increased component of mEPSC frequency was thought to be attributable to Ca²⁺-enhanced events, and, therefore, we used this manipulation to elicit Ca²⁺-enhanced mEPSCs in the following experiments. The increase in mEPSC frequency with 5 mm Ca²⁺_o was not affected by Cd²⁺ (100 μm) or by SKF96365 (100 μm), a broad-spectrum blocker of nonselective cation channels (data not shown). Another possible mechanism to produce this [Ca²⁺]_i increase is contribution of Na⁺/Ca²⁺ exchanger (NCX). NCX is known to exist on the plasma membrane of nerve terminals and may cause net Ca²⁺ influx at the nerve terminal in certain conditions (Mulkey and Zucker, 1992; Blaustein and Lederer, 1999). To test the possible contribution of NCX, we applied an NCX blocker, KB-R7943 (50 μm) or bepridil (50 μm), before changing the [Ca²⁺]_o from 2 to 5 mm. However, neither of these blockers inhibited the increase in mEPSC frequency (data not shown). Therefore, the [Ca²⁺]_i elevation after changing the [Ca²⁺]_o is mediated by mechanisms other than the above tested Ca²⁺ entry pathways.

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Figure 3.

Effect of WIN55,212-2 on mEPSCs with 5 mm Ca²⁺_o. A, Sample traces of mEPSCs recorded at the time points labeled in B. B, MiniatureEPSCselicited by raising [Ca²⁺]_o from 2 to 5 mm were suppressed by WIN55,212-2, and the WIN55,212-2-induced suppression was reversed by SR141716A (2 μm). C, Summary bar graph for the effect of WIN55,212-2 on mEPSC frequency in various experimental conditions. Data are obtained from the indicated number of experiments and normalized to the baseline frequency with 5 mm Ca²⁺_o. **p < 0.01, t test. Error bars represent SEM.

WIN55,212-2 suppresses Ca²⁺-enhanced but VDCC-independent mEPSCs

We examined whether activation of cannabinoid receptors could suppress Ca²⁺-enhanced but VDCC-independent mEPSCs elicited with 5 mm Ca²⁺_o. As exemplified in Figure 3, A and B, bath application of WIN55,212-2 effectively suppressed mEPSC frequency with 5 mm Ca²⁺_o. The mean baseline frequency with 5 mm Ca²⁺_o was 6.2 ± 0.8 Hz (n = 14). WIN55,212-2 reduced the frequency to 21.1 ± 3.8% of the baseline (n = 14; p < 0.01). This suppression was completely reversed by subsequent application of SR141716A (117.5 ± 6.7% of the level before WIN55,212-2 application; n = 14) (Fig. 3A–C). These results indicate that Ca²⁺-enhanced mEPSCs elicited with 5 mm Ca²⁺_o are sensitive to cannabinoids.

Because Ca²⁺ influx through VDCCs is not the primary factor of eliciting Ca²⁺-enhanced mEPSCs with 5 mm Ca²⁺_o, the suppression of mEPSC by WIN55,212-2 is not likely to result from cannabinoid-induced inhibition of VDCCs. This notion was confirmed by our observation that Cd²⁺ (100 μm) had no effect on suppression of mEPSC frequency by WIN55,212-2. In the continuous presence of Cd²⁺, WIN55,212-2 decreased mEPSC frequency to 14.4 ± 3.2% of the baseline (n = 6; p < 0.01) (Fig. 3C). We also examined the possible contribution of 4-AP-sensitive K⁺ channels because their activation is reported to suppress PF–PC synaptic transmission (Daniel and Crepel, 2001). Application of 4-AP (1 mm) with 5 mm Ca²⁺_o had no effect on mEPSC frequency (data not shown). Furthermore, an application of WIN55,212-2 in the continuous presence of 4-AP decreased mEPSC frequency to 20.4 ± 3.1% of the baseline (n = 13; p < 0.01) (Fig. 3C). These results suggest that neither VDCCs nor 4-AP-sensitive K⁺ channels contribute to the cannabinoid-mediated suppression of Ca²⁺-enhanced mEPSC.

Ca²⁺ ionophore-induced mEPSCs are suppressed by WIN55,212-2, whereas α-latrotoxin-induced ones are not

It could be possible that cannabinoid agonists block unknown Ca²⁺ entry pathways and thereby decrease the [Ca²⁺]_i and reduce mEPSC frequency. To test this possibility, we directly raised Ca²⁺ concentration in the presynaptic terminals by using A23187, a Ca²⁺ ionophore. Bath application of A23187 (5 μm) with 2 mm Ca²⁺_o markedly enhanced mEPSC frequency (773.2 ± 58.1% of the baseline; average frequency, 7.9 ± 0.8 Hz; n = 6; p < 0.01). If a cannabinoid agonist acts on the Ca²⁺ entry pathways, it should not affect mEPSCs elicited by A23187. However, subsequent application of WIN55,212-2 effectively suppressed mEPSC frequency to the baseline level before A23187 application (Fig. 4A,B). The suppression induced by WIN55,212-2 was completely reversed by subsequent application of SR141716A (Fig. 4A,B). These results indicate that cannabinoid agonists suppress the Ca²-enhanced mEPSCs by acting on processes downstream of Ca²⁺ entry.

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Figure 4.

Effects of WIN55,212-2 on mEPSCs elicited by application of Ca²⁺ ionophore and α-LTX. A, B, Representative data showing the effect of WIN55,212-2 on mEPSC frequency enhanced by application of a Ca²⁺ ionophore, A23187. A, Sample traces of mEPSCs recorded at the time points labeled in B. B, Miniature EPSCs elicited by A23187 (5 μm) were suppressed by WIN55,212-2 (5 μm), and this suppression was reversed by SR141716A (2 μm). C, D, Representative data showing the effect of WIN55,212-2 on mEPSC frequency enhanced by application of α-LTX. C, Sample traces of mEPSCs recorded at the time points labeled in D. D, WIN55,212-2 (5 μm) and SR141716A (2 μm) had no effect on mEPSC frequency elicited by application of α-LTX.

We then examined whether cannabinoid agonists affect the mEPSCs enhanced independently of [Ca²⁺]_i rise. α-LTX is the major active component of a black widow spider toxin and is known to elicit a high rate of spontaneous transmitter release from nerve terminals (Capogna et al., 1996; Zhou et al., 2000; Harvey and Stephens, 2004). We examined whether cannabinoid agonist interfered with the release machinery activated by α-LTX. Because α-LTX facilitates vesicle release via both Ca²⁺-dependent and Ca²⁺-independent mechanisms (Sudhof, 2001), we used this toxin in the absence of Ca²⁺_o to minimize Ca²⁺ mobilization. Bath application of α-LTX (0.3 nm) produced a marked increase in mEPSC frequency (935.9 ± 103.0% of the baseline; 5.1 ± 0.6 Hz; n = 4). WIN55,212-2 did not affect mEPSC frequency in the presence of α-LTX (106.9 ± 5.6% of the baseline; n = 4; p = 0.82) (Fig. 4C,D). These results indicate that the Ca²⁺-independent release machinery activated by α-LTX is not a target of cannabinoid receptor-mediated signaling.

Figure 5 illustrates the relationship between the effects of WIN55,212-2 on mEPSC frequency and its baseline values in various experimental conditions. WIN55,212-2 markedly inhibited mEPSC frequency in the presence of 5 mm Ca²⁺_o or A23187, when the predominant component of mEPSC was a Ca²⁺-enhanced one. In contrast, WIN55,212-2 did not suppress mEPSCs with 0 or 2 mm Ca²⁺_o, when the Ca²⁺-enhanced mEPSCs were merely occurring. Furthermore, at all concentrations examined (0.01, 0.05, 0.5, and 5 μm), WIN55,212-2 selectively suppressed Ca²⁺-enhanced mEPSCs (supplemental Fig. 1A, available at www.jneurosci.org as supplemental material). The inhibitory effect by WIN55,212-2 is not simply correlated with the frequency of mEPSC because the α-LTX-elicited mEPSCs occurred in high frequency are not affected by WIN55,212-2. Together, these results suggest that activation of cannabinoid receptors selectively suppresses mEPSCs elicited by elevation of the [Ca²⁺]_i, which is produced by a mechanism other than VDCCs (Ca²⁺-enhanced but VDCC-independent mEPSCs).

Basal mIPSCs are not suppressed by WIN55,212-2

We then examined whether basal mIPSCs were sensitive to cannabinoid agonists or not. A previous report shows that prolonged removal of external Ca²⁺ depletes intracellular Ca²⁺ stores and results in reduction of the frequency and amplitude of Ca²⁺-enhanced mIPSCs (Llano et al., 2000). When slices were perfused with a Ca²⁺-free solution containing 200 μm EGTA for >30 min, mIPSC frequency and amplitude markedly decreased. Under this condition, the magnitude of suppression induced by WIN55,212-2 became smaller than that with 2 mm Ca²⁺_o (73.0 ± 4.3% of the baseline; n = 6), although it was still significant (p < 0.05). This is presumably because Ca²⁺-enhanced mIPSC are not completely eliminated even in the absence of Ca²⁺_o (Fig. 2F). Therefore, we performed a similar experiment in the condition in which removal of Ca²⁺_o was combined with loading BAPTA-AM to slices to minimize an elevation in the [Ca²⁺]_i in the presynaptic terminals. Under this condition, WIN55,212-2 did not reduced mIPSC frequency (102.1 ± 6.8% of the baseline; n = 6; p = 0.91) (Fig. 6A,B).

It is reported that ryanodine-sensitive Ca²⁺ stores contribute to the generation of Ca²⁺-enhanced mIPSCs (Llano et al., 2000). Therefore, we tried to eliminate Ca²⁺-enhanced mIPSCs by interfering with ryanodine-sensitive Ca²⁺ stores. After mIPSCs were recorded in normal solution containing 2 mm Ca²⁺_o, the slice was perfused for 15 min with a high-K⁺ solution (15 mm) supplemented with 100 μm ryanodine. Then the slice was perfused for another 15 min with normal external solution (2.5 mm K⁺) supplemented with ryanodine. After this treatment, both mIPSC frequency and mean amplitude were markedly reduced from those before ryanodine application (2.1 ± 0.5 Hz and 45.7 ± 8.0 pA compared with 4.0 ± 0.4 Hz and 59.0 ± 12.3 pA in control period; n = 6; p < 0.01). These changes were not observed after the challenge of high-K⁺ solution (15 mm) without ryanodine (data not shown). We then compared the depressant effect of WIN55,212-2 in these two groups. WIN55,212-2 reduced mIPSC frequency to 45.2 ± 8.9% after the control manipulation without ryanodine, whereas the effect was significantly smaller after the manipulation with ryanodine (69.5 ± 4.2%; n = 6; p < 0.01) (Fig. 6C,D).

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Figure 5.

Summary plots for the effects of WIN55,212-2 on the mEPSC frequency in various experimental conditions. Values on the ordinate are expressed relative to the baseline mEPSC frequency before application of WIN55,212-2 (5 μm). Each symbol represents mean ± SEM. White rectangle, 0 mm Ca²⁺_o (n = 8); light gray rectangle, 2 mm Ca²⁺_o (n = 5); black rectangle, 5 mm Ca²⁺_o (n = 14); black triangle, A23187 (n = 6); white circle, 0 mm Ca²⁺_o plus α-LTX (n = 4).

Together, the results from the two sets of experiments strongly suggest that mIPSCs become insensitive to cannabinoid agonists when generation of Ca²⁺-enhanced mIPSCs is suppressed.

WIN55,212-2 does not interfere with the α-LTX-activated release process

We then examined whether cannabinoid agonists affect Ca²⁺-independent enhancement of mIPSCs by α-LTX. To minimize Ca²⁺-enhanced mIPSCs, application of EGTA-containing Ca²⁺-free solution was combined with presynaptic loading of BAPTA-AM, and then α-LTX was applied with Ca²⁺-free solution. α-LTX significantly increased mIPSC frequency (1672.3 ± 198.8% of the control; 15.4 ± 1.9 Hz; n = 4), but the α-LTX-induced mIPSCs were not suppressed by WIN55,212-2 (96.4 ± 3.5% of the baseline; n = 4; p = 0.71) (Fig. 6E,F). These results indicate that Ca²⁺-independent mIPSCs activated by α-LTX are insensitive to cannabinoid agonists.

As shown in Figure 7, in the experiments changing the [Ca²⁺]_o, the magnitude of mIPSC suppression by WIN55,212-2 was correlated to the baseline mIPSC frequency that reflected the proportion of Ca²⁺-enhanced mIPSC. In contrast, WIN55,212-2 did not affect Ca²⁺-independent mIPSCs elicited by α-LTX. We also examined the depressant effect of WIN55,212-2 at 0.01, 0.05, 0.5, and 5 μm. At all concentrations tested, WIN55,212-2 selectively suppressed Ca²⁺-enhanced mIPSCs (supplemental Fig. 1B, available at www.jneurosci.org as supplemental material).

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Figure 6.

Effects of WIN55,212-2 on basal and α-LTX-induced mIPSCs. A, B, Representative data showing the effect of WIN55,212-2 on mIPSC frequency with EGTA-containing 0 mm Ca²⁺_o solution preincubated with BAPTA-AM. A, Sample traces of mIPSCs at the time points labeled in B. B, WIN55,212-2 (5μm) and SR141716A (2μm) had no effect on mIPSC frequency. C, D, Representative data showing the effect of WIN55,212-2 when Ca²⁺-enhanced mIPSC is suppressed by 100μm ryanodine. C, Sample traces of mIPSCs at the time points labeled in D. D, The depressant effect of WIN55,212-2 (5 μm) is not prominent compared with the example shown in Figure 1 D. E, F, Representative data showing the effect of WIN55,212-2 on the frequency of α-LTX-induced mIPSCs. To eliminate Ca²⁺-enhanced mIPSC, application of 0 mm Ca²⁺_o solution containing EGTA (200 μm) was combined with presynaptic loading of BAPTAAM. E, Sample traces of mIPSCs at the time points labeled in F. F, WIN55,212-2 (5 μm) and SR141716A (2 μm) had no effect on mIPSC frequency enhanced by application of α-LTX.

These results strongly suggest that the mIPSCs independent of spontaneous [Ca²⁺]_i rise are not suppressed by cannabinoid agonists.

Baclofen and l-AP-4 selectively suppress Ca²⁺-enhanced miniature events

Finally, we tested whether selective suppression of miniature events was commonly seen for other G_i/o-coupled receptor-mediated presynaptic inhibition. PF to PC excitatory synaptic transmission is known to be suppressed presynaptically by activating GABA_B receptors or group III mGluRs (Dittman and Regehr, 1996; Pekhletski et al., 1996). In the presence of 2 mm Ca²⁺_o, the mEPSC frequency was not affected by baclofen (5 μm), a GABA_B receptor agonist, or l-AP-4 (50 μm), a group III mGluR agonist (normalized mEPSC frequency was 96.2 ± 3.0% with baclofen, n = 12, p = 0.61; 106.7 ± 7.1% with l-AP-4, n = 6, p = 0.72) (Fig. 8A,B). In contrast, in the presence of 5 mm Ca²⁺_o, the mEPSC frequency was markedly suppressed by baclofen and l-AP-4 (normalized mEPSC frequency was 16.3 ± 4.0% with baclofen, n = 6, p < 0.01; 19.1 ± 2.1% with l-AP-4, n = 18, p < 0.01) (Fig. 8A,B). These results indicate that activation of presynaptic GABA_B receptor or group III mGluR preferentially suppresses Ca²⁺-enhanced mEPSCs.

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Figure 7.

Summary plots for the effects of WIN55,212-2 on mIPSC frequency in various experimental conditions. Values on the ordinate are expressed relative to the baseline mIPSC frequency before application of WIN55,212-2 (5 μm). Each symbol represents mean ± SEM. White rectangle, 0 mm Ca²⁺_o plus BAPTA-AM (n = 6); light gray rectangle, 0 mm Ca²⁺_o (n = 6); gray circle, 2 mm Ca²⁺_o plus 100 μm ryanodine (n = 6); gray rectangle, 2 mm Ca²⁺_o (n = 9); black rectangle, 5 mm Ca²⁺_o (n = 6); white circle, 0 mm Ca²⁺_o plus BAPTA-AM plusα-LTX (n = 4).

Previous studies show that baclofen effectively suppresses mIPSC frequency in the presence of 2 mm Ca²⁺_o (Than and Szabo, 2002; Harvey and Stephens, 2004). To test whether this suppression is selective for Ca²⁺-enhanced mIPSCs, we combined loading of BAPTA-AM and removal of external Ca²⁺ to minimize Ca²⁺-enhanced mIPSCs (Fig. 8D). Under this condition, the magnitude of baclofen-induced suppression in a Ca²⁺-free, EGTA-containing solution was significantly smaller than that in a 2 mm Ca²⁺_o solution (normalized mIPSC frequency with baclofen was 69.0 ± 8.0% in 0 mm Ca²⁺_o plus BAPTA-AM, n = 6; 32.9 ± 2.9% in 2 mm Ca²⁺_o solution, n = 6; p < 0.01) (Fig. 8D). These results indicate that GABA_B receptor activation also preferentially suppresses Ca²⁺-enhanced mIPSCs. However, in contrast to the cannabinoid agonists, baclofen produces small but significant suppression of mIPSC frequency even when an application of Ca²⁺-free solution is combined with BAPTA-AM loading.

Heterogeneity of miniature events and cannabinoid sensitivity

In PCs, mIPSC is suppressed by cannabinoid agonists, whereas mEPSC is not at the normal [Ca²⁺]_o (Takahashi and Linden, 2000). This result has been regarded as evidence that release machinery is a target for presynaptic depression of the inhibitory inputs but not for that of the excitatory ones. However, our results indicate that mEPSCs become sensitive to cannabinoid agonists when the occurrence of Ca²⁺-enhanced mEPSCs is facilitated by raising the [Ca²⁺]_i in the presynaptic terminals (Fig. 5). Conversely, mIPSCs became insensitive to cannabinoid agonists when the occurrence of Ca²⁺-enhanced mIPSCs was suppressed (Fig. 7). These results suggest that, at least in PCs, the cannabinoid sensitivity of miniature events depends on the proportion of Ca²⁺-enhanced component in the total miniature events. Therefore, the aforementioned interpretation that the apparent cannabinoid insensitivity excludes possible inhibitory effect on release machinery is not the case at least for PC synapses.

In the hippocampus, it has also been reported that the cannabinoid sensitivity of miniature synaptic events changes depending on recording conditions (Hoffman and Lupica, 2000; Wilson and Nicoll, 2001; Varma et al., 2002). Miniature mIPSCs of hippocampal pyramidal neurons are normally insensitive to WIN55,212-2. However, mIPSCs facilitated by a high external K⁺ concentration are suppressed by WIN55,212-2 (Hoffman and Lupica, 2000; Wilson and Nicoll, 2001; Varma et al., 2002). Because the mIPSC facilitation triggered by a high external K⁺ concentration is also suppressed by Cd²⁺, the above results have been taken as evidence that VDCCs are the main target of the cannabinoid-mediated presynaptic depression. However, because Cd²⁺ blocks not only VDCCs themselves but also the subsequent Ca²⁺ influx and enhancement of vesicle release, the experiment with Cd²⁺ is inconclusive. In the present study, mEPSCs triggered by [Ca²⁺]_i rise were suppressed by WIN55,212-2 (Fig. 5). Therefore, if mIPSCs of hippocampal pyramidal cells have similar properties to the mEPSCs of cerebellar PCs, the possibility remains that cannabinoid agonists inhibit the Ca²⁺-enhancd mIPSCs elicited by Ca²⁺ influx through VDCCs. Moreover, it has been shown that mIPSCs recorded in layer 2/3 pyramidal neurons in the cerebral cortex are generally sensitive to WIN55,212-2, but they become insensitive when VDCC or subsequent Ca²⁺ influx is blocked (Trettel and Levine, 2002). These data can also be interpreted in a similar way that cannabinoid agonists inhibit Ca²⁺-enhanced mIPSCs.

Suppression of miniature events by activation of GABA_B receptor or group III mGluR

In the present study, baclofen and l-AP-4 suppressed mEPSC frequency with 5 mm Ca²⁺_o but not with 2 mm Ca²⁺_o. This indicates that activation of GABA_B receptor or group III mGluR of PFs also has a tendency to suppress mEPSCs triggered by [Ca²⁺]_i rise. GABA_B receptor, group III mGluR, and cannabinoid receptor are all coupled to G_i/o-protein and may share common signaling cascades for presynaptic depression in PF terminals. However, a previous report has shown that mEPSCs recorded in PCs can be suppressed by baclofen (Dittman and Regehr, 1996). This apparent discrepancy from our results may be attributed to species difference (between rat and mouse) or to the differential incidence of Ca²⁺-enhanced events at the normal [Ca²⁺]_o. Because a previous study (Dittman and Regehr, 1996) reports an mEPSC frequency (∼3 Hz) that is significantly higher than our data (∼0.5 Hz), we assume that a considerable portion of mEPSCs recorded in their experiment might be Ca²⁺-enhanced events and therefore sensitive to baclofen.

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Figure 8.

Effects of baclofen and l-AP-4 on miniature synaptic events. A, Representative data showing the effect of baclofen on frequency of mEPSCs. Baclofen had no effect with 2 mm Ca²⁺_o but suppressed mEPSC frequency with 5 mm Ca²⁺_o. B, Summary bar graph showing the effect of baclofen and l-AP-4 with 2 and 5 mm Ca²⁺_o. Data are normalized to the baseline frequency before application of baclofen or l-AP-4 and shown as mean ± SEM for the indicated number of experiments. **p < 0.01, t test. C, Representative data showing the effect of baclofen on frequency of mIPSCs. Baclofen markedly suppressed mIPSC frequency with 2 mm Ca²⁺_o. The effect of baclofen was much weaker than that with 0 mm Ca²⁺_o plusBAPTA-AM. D, Summary bar graph showing the effect of baclofen with 2 mm Ca²⁺_o (n = 6) and with 0 mm Ca²⁺_o plusBAPTA-AM (n=6). Data are normalized to the baseline frequency before application of baclofen and shown as mean ± SEM for the indicated number of experiments. **p < 0.01, t test.

We showed that mIPSC frequency was suppressed by baclofen in the presence of 2 mm Ca²⁺_o, and the depressant effect was significantly reduced by Ca²⁺ removal together with BAPTA-AM loading (Fig. 8C,D). This indicates that baclofen preferentially suppresses Ca²⁺-enhanced mIPSCs. Previous reports have shown that the effect of baclofen on mIPSCs is not affected by Cd²⁺ (Than and Szabo, 2002; Harvey and Stephens, 2004), indicating that VDCCs are not involved in the baclofen-induced suppression of mIPSCs. Importantly, in PCs, the mIPSCs facilitated by ionomycin are suppressed by baclofen (Than and Szabo, 2002), whereas those triggered by α-LTX are not (Harvey and Stephens, 2004). Together, the mIPSC depression by GABA_B receptor activation may have common nature with the cannabinoid-mediated one. However, a recent report demonstrates that perfusion with a Ca²⁺-free solution or presynaptic loading of BAPTA-AM does not influence the effect of baclofen on mIPSCs in PCs (Harvey and Stephens, 2004). A possible reason for this apparent discrepancy from our result is imperfect blockade of Ca²⁺-enhanced miniature events. We found that BAPTA-AM loading alone was not sufficient to suppress Ca²⁺-enhanced mIPSCs because the subsequent [Ca²⁺]_o removal further suppressed mIPSC frequency.

Baclofen appeared to have a slight effect on mIPSCs even after Ca²⁺_o removal combined with BAPTA-AM loading. This might be because either baclofen suppresses the basal mIPSCs or the Ca²⁺-enhanced mIPSCs still remain under such condition. Then, why does Ca²⁺_o removal exert different effects after GABA_B receptor and cannabinoid receptor activation? We found that the depressant effect of baclofen on mIPSCs was stronger than that of WIN55,212-2 in the presence of 2 mm Ca²⁺_o (32.9 ± 2.9% with baclofen; 56.9 ± 5.2% with WIN55,212-2) (Figs. 7, 8C,D). Therefore, it could be possible that coupling efficacy of GABA_B receptor to the GABA release mechanism is stronger than that of cannabinoid receptor to the release mechanism.

Possible contribution to synaptic transmission

In the cerebellum, basket cell to PC transmission is partially mediated by Ca²⁺ release from intracellular stores (Bardo et al., 2002; Galante and Marty, 2003). In addition, mIPSCs with 2 mm Ca²⁺_o are also mediated by Ca²⁺ release from internal stores (Llano et al., 2000). In contrast, Ca²⁺ release does not contribute to evoked or miniature excitatory synaptic responses in PCs (Carter et al., 2002). In contrast, in the hippocampus, Ca²⁺ release is reported to contribute to excitatory but not to inhibitory transmission (Emptage et al., 2001; Bouchard et al., 2003). Several previous reports have consistently shown that, at normal external Ca²⁺ and K⁺ concentrations, mIPSCs in the hippocampus are not affected by cannabinoid agonists or endocannabinoids released by DSI protocol (Pitler and Alger, 1994; Alger et al., 1996; Hajos et al., 2000), whereas mEPSCs are clearly suppressed (Misner and Sullivan, 1999; Sullivan, 1999). Therefore, it is conceivable that, at the normal [Ca²⁺]_o, mEPSCs in the hippocampus may involve Ca²⁺-enhanced events and thus sensitive to cannabinoid agonists, whereas mIPSCs may be almost devoid of Ca²⁺-enhanced events and thus insensitive to cannabinoid agonists. Together, these lines of evidence suggest that Ca²⁺-enhanced miniature synaptic events are specifically suppressed by activation of cannabinoid receptors and presumably also of other G_i/o-coupled receptors in various brain regions.

Because synaptic transmission by an action potential is triggered by rapid [Ca²⁺]_i rise in the presynaptic terminals through VDCCs, released vesicles are regarded as Ca²⁺-dependent ones. Therefore, certain extent of presynaptic depression of action potential-evoked transmitter release by G_i/o-coupled receptors may be attributed to the suppression of Ca²⁺-dependent release machinery. Activation of G_i/o-coupled receptors also reduces Ca²⁺ influx into presynaptic terminals presumably through inhibition of VDCCs (Dittman and Regehr, 1996; Diana et al., 2002; Brown et al., 2004), which can also contribute to the suppression of transmitter release.

A previous report suggests that presynaptic depression by cannabinoid agonist is mainly induced by suppression of Ca²⁺ influx at the PF–PC synapse (Brown et al., 2004). In contrast, presynaptic depression by baclofen at the same synapse is induced by both reduced Ca²⁺ influx and inhibition of release machinery (Dittman and Regehr, 1996). These results suggest that the contribution of the modulation of Ca²⁺-dependent release machinery to presynaptic depression may vary depending on the classes of G_i/o-coupled receptors. At the inhibitory synapses of PCs, Diana and Marty (2003) estimated that reduction of mIPSC frequency contributed to 13.4% of DSI. In addition, mIPSC amounted to ∼47% of spontaneous IPSC in PCs (Diana and Marty, 2003). Therefore, modulation of Ca²⁺-enhanced mIPSCs seems to have important roles in controlling inhibitory synaptic transmission to PCs. Because Ca²⁺-enhanced miniature events are present at synapses in various brain regions (Llano et al., 2000; Bardo et al., 2002; Simkus and Stricker, 2002), specific suppression of these events by cannabinoid receptor and other G_i/o-coupled receptors may be an important mechanism for modulating synaptic transmission.