140 W Chang, et al receptor activation (Ca2＋-assisted RER, Ca-RER) and acts presynaptically to suppress neurotransmitter release. Unlike DSE/DSI, Ca-RER can be induced by weak elevation of calcium, which can be mediated by calcium sources other than the voltage-operated calcium channel (VOCC) like the Endoplasmic reticulum (ER) calcium store or TRPC. It was reported that ER calcium release do not have a role in the induction of Ca-RER [22], indicating it is more worthwhile to investigate the role of TRPC in endocannabinoid release. Here, we investigated whether endocannabinoid-induced retrograde signaling is regulated by the TRPC-induced slow current. We induced transient depression of synaptic trans- mission by PF burst stimulation in current clamp mode and associative stimulation of parallel fiber (PF) burst and Purkinje cell (PC) depolarization in voltage clamp mode; however, neither was affected by TRPC blockers. This sug- gests TRPC mediated slow currents and calcium transients do not play an important role in the endocannabinoid sig- naling and that calcium sources other than TRPC is needed for Ca-RER. METHODS Parasagittal brain slices of the cerebellar vermis (250 μm thick) were prepared from P15-P20 Sprague-Dawley rats using a vibrating tissue slicer and ice-cold standard artifi- cial cerebrospinal fluid (ACSF) containing 124 mM NaCl, 2.5 mM KCl, 1 mM Na2HPO4, 1.3 mM MgCl2, 2.5 mM CaCl2, 26.2 mM NaHCO3, and 20 mM D-glucose, bubbled with 95% O2 and 5% CO2. After cutting, slices were kept for 30 min at 35oC and then for up to 8 hrs. at 25oC in ACSF. After a recovery period, the slices were placed in a sub- merged chamber that was perfused at a rate of 2 ml/min with ACSF supplemented with either 100 μM picrotoxin to block GABAA receptors. Somatic whole-cell recording were obtained by using an EPC8 amplifier (HEKA Instruments Inc., Bellmore, NY). The recording electrodes (resistance 2∼4 MΩ) were filled with a solution containing 130 mM K-gluconate, 10 mM KCl, 10 mM HEPES, 0.2 EGTA, 10 mM Na2-phosphocreatine, 3 mM Mg2ATP, 0.3 mM Na3GTP (pH 7.25) for current-clamp recording and 135 mM Cs-methanesulfonate, 10 mM CsCl, 10 mM HEPES, 4 mM Mg2ATP, 0.4 mM Na3GTP, and 0.2 mM EGTA (pH 7.25) for voltage-clamp recordings. Currents were filtered at 1 kHz, digitized at 5 kHz, and acquired using Pulse (HEKA Instruments). For parallel fiber stimulation, stand- ard patch pipettes were used that were filled with external saline and placed in the middle third of the molecular layer. Synaptic responses were evoked every 30 s using 20∼40 μA pulses (100 μs duration). When burst stimulation was employed, the interpulse interval was 10 ms. Recordings were performed at RT in ACSF. All group data are shown as mean±SEM. Comparisons were made using Student's t tests. All drugs were pur- chased from Sigma (St. Louis, MO) except for CPCCOEt, NBQX, AM251, SKF96365 (Tocris Cookson, Ballwin, MO). RESULTS Several studies demonstrated that brief burst of parallel fiber can trigger retrograde inhibition of the PF-PC synapse [16,22]. This phenomenon is termed as Ca-RER and is Fig. 1. PF burst induced Endocannabinoid-mediated short-term depression in cerebellar Purkinje cell. (A) The induction protocol for transient depression and trace in induction. 10 parallel fiber stimuli were applied at 100 Hz in current clamp mode. (B) EPSPs before induction and 5 s after induction. (Top: control, bottom: 3 μM of AM251 applied, Black: before induction, Grey: 5 s after induction) (C) 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, 3 μM of AM251 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 AM251. (D) Summary bar graphs showing change of depression of EPSPs after application of AM251. mGluR-PLCβ4 dependent. To mimic this Ca-RER, we made whole-cell patch clamp recordings from cerebellar Purkinje cells in cerebellar slices and recorded parallel fi- ber-Purkinje cell (PF-PC) excitatory postsynaptic potentials (EPSPs) every 2 s, then applied short burst PF stimulation (100 Hz×10) (Fig. 1A). Subsequent EPSCs were depressed transiently (Fig. 1B, C, D; 52.05±10.57%, n=5), and this short-term depression was inhibited by 3 μM of cannabi- noid receptor 1 blocker AM251 (Fig. 1B, C, D; −23.69± 15.20%, n=5). Furthermore, in another set of experiments, short- term depression (Fig. 2; 49.73±25.25%, n=5) was also inhibited by 100 μM of CPCCOEt, an mGluR inhibitor (Fig. 2; −12.37±17.65%, n=5). This data shows that the de- pression induced by burst stimulation of PF is CB1R and mGluR1-dependent RER. TRPC is known to mediate slow inward currents acti- vated by mGluR1 in cerebellar Purkinje cells [8,23]. TRPC is a calcium-permeable non-selective cation channel, thus it is possible that TRPC functions as a calcium source in calcium-dependent cellular events. To investigate whether TRPC-mediated calcium influx contributes to Ca-RER, we applied SKF96365, a TRP channel inhibitor. The short- term depression (Fig. 3; 38.42±11.10%, n=5) was inhibited by 30 μM of SKF96365 (Fig. 3; −13.62±4.99%) signifi- cantly. Regehr and his coworkers reported that associative stimulation can induce endocannabinoid signaling more ef- fectively [22]. It is suggested that PF burst can cause a mas- sive release of glutamate which can activate mGluR1 on the perisynaptic site of Purkinje cell dendritic spines, and climbing fiber (CF) can make global depolarization of Purkinje cell that may open VOCC, increasing the intra-