142 W Chang, et al Fig. 4. TRP blocker SKF96365 inhibited endocannabinoid-mediated short-term depression induced by association of PF burst and PC depolarization. (A) The induction protocol for transient depression and current trace in induction. 10 parallel fiber stimuli at 100 Hz and 50 ms of PC depolarization 50 ms after PF burst were applied in voltage clamp mode. (B) EPSCs before induction and 5 s after induction (Left: control, right: 30 μM of SKF96365 applied, Black: before induction, Grey: 5 s after induction). (C) EPSCs were evoked by a paired pulse of PF stimulus during a 40 seconds baseline and for 60 seconds after induction of transient depression. After that, 30 μM of SKF96365 was applied for 10 minutes, and then record- ing of baseline and induction of transient depression were established in the same cell. Amplitudes of EPSCs were normalized to baseline before and after application of SKF96365. (D) Summary bar graphs showing change of depression of EPSCs after appli- cation of SKF96365. most of the slow current. This means the action of SKF96365 was a nonspecific effect, although the mecha- nism by which SKF96365 inhibited RER is unknown. Drugs like SKF96365 or ruthenium red have been used as TRP channel blockers so far, but now they are known to have nonspecific effects on Endoplasmic reticulum (ER) calcium stores or channels other than TRPC. BTP2 has been known to function as a store-operated Ca2＋ channel/Ca2＋ re- lease-activated Ca2＋ channel blocker [34,35], and inhibit overexpressed TRPC channels in HEK293 cells. BTP2 al- most fully blocks TRPC at about 1 μM, and had little effect on ER, K＋ channel or VOCC in cultured single cell. However, a concentration of BTP2 that blocks TRPC effec- tively in brain slices has not been reported yet. We found that 50 μM of BTP2 is needed to block the slow current; it is much larger than the concentration that blocks TRPC in cultured single cell, and specificity of BTP2 to TRPC at this concentration is not clear yet. However, based on the fact that BTP2 did not affect endocannabinoid release, we conclude that TRPC does not contribute to endocannabinoid release although 50 μM of BTP2 may be nonspecific. The results described above suggest that BTP2 can be an alter- native or additional option as a pharmacologic blocker of TRP channels. The role of VOCC or ER calcium stores as a calcium source is well known, while that of calcium influx through TRPC is unclear. We investigated a possibility that TRPC plays a role as a novel calcium source other than VOCC or ER calcium store in Ca-RER, however, TRPC did not Fig. 5. Endocannabinoid-mediated short-term depression in cere- bellar Purkinje cell was not inhibited by BTP2. (A) Slow current traces evoked by 10 PF stimuli at 100 Hz during 5 μM of NBQX (Black: Control, Grey: 50 μM of BTP2). (B) Slow EPSCs were evoked during a 200 seconds baseline and for 500 seconds after induction of transient depression application. After that, 50 μM of BTP2 was applied for 10 minutes, and then recording of baseline and induction of transient depression were established in the same cell. (C) EPSPs were evoked by paired pulse of PF stimulus during a 40 seconds baseline and for 60 seconds after induction of transient depression. After that, 50 μM of BTP2 was applied for 10 minutes, and then recording of baseline and induction of transient depression were established in the same cell. Amplitudes of EPSCs were normalized to baseline before and after application of BTP2. (D) Summary bar graphs showing change of depression of EPSPs after application of BTP2. (E) EPSPs were evoked by paired pulse of PF stimulus during a 40 seconds baseline and for 60 seconds after induction of transient depression. After that, 50 μM of BTP2 was applied for 10 minutes, and then recording of baseline and induction of transient depression were established in the same cell. Amplitudes of EPSCs were normalized to baseline before and after application of BTP2. (F) Summary bar graphs showing change of depression of EPSCs after application of BTP2. affect endocannabinoid release. It was also revealed pre- viously that ER calcium store has little effect on the ele- vation of calcium concentration and endocannabinoid re- lease [22]. Based on these findings, there is a large possi- bility that VOCC acts as the critical and sole calcium source. We could not induce transient depression by only PF burst on voltage clamp mode at −70 mV, unlike associa- tive stimulation or PF burst on current clamp mode, which VOCC can activate by; this data is consistent with the idea that VOCC is the most important calcium source for endo- cannabinoid release.