Effects of TRPC Blockers on eCB Signaling 141 Fig. 2. PF burst-induced Endocannabinoid-mediated short-term depression is mGluR-dependent. (A) EPSPs before induction and 5s after induction (Top: control, bottom: 100 μM of CPCCOEt applied, Black: before induction, Grey: 5 s after induction). (B) EPSPs were evoked by a single pulse of PF stimulus during an 80 seconds baseline and for 60 seconds after induction of transient depression. After that, 100 μM of CPCCOEt was applied for 10 minutes, and then recording of baseline and induction of transient depression were established in the same cell. Amplitudes of EPSPs were normalized to baseline before and after application of CPCCOEt. (C) Summary bar graphs showing change of depression of EPSPs after application of CPCCOEt. cellular calcium concentration of the Purkinje cell. To mim- ic this associative short-term depression and investigate the role of TRPC in the depression, we made whole-cell patch clamp recordings from cerebellar Purkinje cells in cerebellar slices and recorded PF-PC EPSCs, then applied associative stimulation consisting of short burst PF stim- ulation (100 Hz×10) followed by 50 ms of depolarization of PC after 50 ms (Fig. 4A). The associative protocol induced short-term depression effectively (Fig. 4B, C, D; 50.03± 14.84%, n=7), and SKF96365 decreased this depression par- tially (Fig. 4B, C, D; 14.69±11.72%, n=7). Although we observed the effect of a TRP channel blocker, we had to confirm that TRPC specifically contributed to short-term plasticity since SKF96365 is known for its non-specificity [24,25], especially to VOCCs. To investigate whether TRPC-mediated calcium influx contribute to RER, we applied BTP2, another TRPC inhibitor. 1 μM of BTP2 blocked most TRPC-induced currents in cultured cells in previous studies [26], however, the concentration of BTP2 to block TRPC effectively in slices has not been reported. To find out the optimum concentration of BTP2 to block slow currents in brain slices, we induced TRPC-mediated slow current with parallel fiber burst (100 Hz×10 times) while applying NBQX (2.5 μM) to block the overlaying AMPAR current (Fig. 5A), and examined the concen- tration-dependent effect of BTP2 on the amplitude of the slow current. 50 μM of BTP2 blocked the slow current in slice, while 25 μM or lower concentrations of BTP2 could Fig. 3. TRP blocker SKF96365 inhibited PF burst-induced Endocannabinoid-mediated short-term depression. (A) EPSPs before induction and 5 s after induction (Top: control, bottom: 30 μM of SKF96365 applied, Black: before induction, Grey: 5 s after induction). (B) EPSPs were evoked by a single pulse of PF stimulus during an 80 seconds baseline and for 60 seconds after induction of transient depression. After that, 30 μM of SKF96365 was applied for 10 minutes, then recording of baseline and induction of transient depression were established in the same cell. Amplitudes of EPSPs were normalized to baseline before and after application of SKF96365. (C) Summary bar graphs showing change of depression of EPSPs after application of SKF96365. not (Fig. 5B). Thus, we investigated whether 50 μM of BTP2 changed the amplitude of transient depression of EPSC in cerebellar slices. In cells which showed the depres- sion by 10 times of parallel fiber burst on current clamp mode, 50 μM of BTP2 did not affect the amplitude of tran- sient depression (Fig. 5C, D), although it was enough to block TRPC-mediated slow current almost completely. Furthermore, BTP2 did not affect short-term depression in- duced by the associative protocol in the voltage clamp mode (Fig. 5E, F). This data indicates that TRPC-mediated slow current does not contribute to RER by the associative proto- col we used and that the calcium influx through TRPC is overwhelmed by the calcium influx through VOCC or cal- cium release from intracellular calcium stores. DISCUSSION SKF96365 has been reported to elevate intracellular cal- cium ion levels [27] and inhibit VOCCs [24,28] ? especially T-type calcium channels in cerebellar Purkinje cells [25], potassium channels [29,30], cytochrome P450 [31,32], and calcium ATPase [28,33]. SKF96365 blocked the endocanna- binoid-induced depression effectively; we assumed the SKF96365 inhibited endocannabinoid release by reducing postsynaptic calcium increase through TRPC. However, BTP2, another blocker of TRP channel did not block RER, even though same concentration of BTP2 could block the