Korean J Physiol Pharmacol.  2012 Apr;16(2):139-144. 10.4196/kjpp.2012.16.2.139.

TRPC-Mediated Current Is Not Involved in Endocannabinoid-Induced Short-Term Depression in Cerebellum

• Chang W
• Park JM
• Kim J
• Kim SJ

• KMID: 2071760
• DOI: http://doi.org/10.4196/kjpp.2012.16.2.139

Abstract

It has been reported that activation of metabotropic glutamate receptor 1 (mGluR1) can mediate endocannabinoid-induced short-term depression of synaptic transmission in cerebellar parallel fiber (PF)-Purkinje cell (PC) synapse. mGluR1 has signaling pathways involved in intracellular calcium increase which may contribute to endocannabinoid release. Two major mGluR1-evoked calcium signaling pathways are known: (1) slow-kinetic inward current carried by transient receptor potential canonical (TRPC) channel which is permeable to Ca2+; (2) IP3-induced calcium release from intracellular calcium store. However, it is unclear how much each calcium source contributes to endocannabinoid signaling. Here, we investigated whether calcium influx through mGluR1-evoked TRPC channel contributes to endocannabinoid signaling in cerebellar Purkinje cells. At first, we applied SKF96365 to inhibit TRPC, which blocked endocannabinoid-induced short-term depression completely. However, an alternative TRP channel inhibitor, BTP2 did not affect endocannabinoid-induced short-term depression although it blocked mGluR1-evoked TRPC currents. Endocannabinoid signaling occurred normally even though the TRPC current was mostly blocked by BTP2. Our data imply that TRPC current does not play an important role in endocannabinoid signaling. We also suggest precaution in applying SKF96365 to inhibit TRP channels and propose BTP2 as an alternative TRPC inhibitor.

Keyword

Endocannabinoid; TRPC; Short-term depression; Cerebellar Purkinje cell; BTP2

MeSH Terms

Calcium
Calcium Signaling
Cerebellum
Depression
Endocannabinoids
Imidazoles
Purkinje Cells
Receptors, Metabotropic Glutamate
Synapses
Synaptic Transmission
Calcium
Endocannabinoids
Imidazoles
Receptors, Metabotropic Glutamate

Figure

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  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.

• 

  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.

• 

  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.

• 

  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 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 SKF96365. (D) Summary bar graphs showing change of depression of EPSCs after application of SKF96365.

• 

  Fig. 5 Endocannabinoid-mediated short-term depression in cerebellar 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.

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