Agonist‐Evoked Ca²⁺ Mobilization from Stores Expressing Inositol 1,4,5‐Trisphosphate Receptors and Ryanodine Receptors in Cerebellar Granule Neurones

Abstract

The mechanisms involved in Ca2+ mobilization evoked by the muscarinic cholinoceptor (mAChR) agonist carbachol (CCh) and N‐methyl‐d‐aspartate (NMDA) in cerebellar granule cells have been investigated. An initial challenge with caffeine greatly reduced the subsequent intracellular Ca2+ concentration ([Ca2+]i) response to CCh (to 45 ± 19% of the control), and, similarly, a much reduced caffeine response was detectable after prior stimulation with CCh (to 27 ± 6% of the control). CCh‐evoked [Ca2+]i responses were inhibited by preincubation with thapsigargin (10 µM), 2,5‐di(tert‐butyl)‐1,4‐benzohydroquinone (BHQ; 25 µM), ryanodine (10 µM), or dantrolene (25 µM). BHQ pretreatment was found to have no effect on the sustained phase of the NMDA‐evoked [Ca2+]i response. Both CCh (1 mM) and 1‐aminocyclopentane‐1S,3R‐dicarboxylic acid (ACPD; 200 µM) evoked a much diminished increase in [Ca2+]i in granule cells pretreated with CCh for 24 h compared with vehicle‐treated control cells (CCh, 23 ± 14%; ACPD, 27 ± 1% of respective control values). In contrast, a 24‐h CCh pretreatment decreased the subsequent inositol 1,4,5‐trisphosphate (InsP3) response to CCh to a much greater extent compared with responses evoked by metabotropic glutamate receptor (mGluR) agonists; this suggests that the former effect on Ca2+ mobilization represents a heterologous desensitization of the mGluR‐mediated response distal to the pathway second messenger. Furthermore, [Ca2+]i responses to caffeine and NMDA were unaffected by a 24‐h pretreatment with CCh. This study indicates that ryanodine receptors, as well as InsP3 receptors, appear to be crucial to the mAChR‐mediated [Ca2+]i response in granule cells. As BHQ apparently differentiates between the CCh‐ and NMDA‐evoked responses, it is possible that the directly InsP3‐sensitive pool is physically different from the ryanodine receptor pool. Also, activation of InsP3 receptors may not contribute significantly to NMDA‐evoked elevation of [Ca2+]i in cerebellar granule cells. A model for the topographic organization of cerebellar granule cell Ca2+ stores is proposed.

Intracellular Calcium Regulation by Burst Discharge Determines Bidirectional Long-Term Synaptic Plasticity at the Cerebellum Input Stage

“…Ca 2ϩ entry through VDCCs, by signaling membrane depolarization and repetitive spike discharge (Armano et al, 2000), could also favor mf-GrC LTP. Ca 2ϩ signals were remarkably amplified and protracted by Ca 2ϩ -induced Ca 2ϩ release (CICR), as indicated by the effect of thapsigargin (and also observed in GrC culture) (Irving et al, 1992a,b;Simpson et al, 1996), suggesting that CICR plays a critical role in the induction of long-term plasticity (Nishiyama et al, 2000) at the mf-GrC synapse. It should be noted that GrC [Ca 2ϩ ] i regulatory mechanisms could be differently engaged, depending on mf-GrC operating conditions.…”

“…Moreover, although previous observations suggested the involvement of Ca 2ϩ -dependent mechanisms, these have never been investigated. Reports in cell culture have revealed Ca 2ϩ entering through NMDA and VDCCs and released by inositol 1,4,5-trisphosphate (IP 3 ) and ryanodinsensitive intracellular Ca 2ϩ stores (Irving et al, 1992a,b;Simpson et al, 1996;del Rio et al, 1999;Masgrau et al, 2001;Monti et al, 2002); however, because [Ca 2ϩ ] i changes were stimulated by exogenous drug application and measured in the soma, neither their input specificity nor their relationship with input patterns or synaptic plasticity could be assessed.…”

Ryanodine receptors are involved in muscarinic antinociception in mice

“…It should also be noted that an increased intracellular calcium mobilization through RyR activation has been demonstrated after stimulation of muscarinic receptors. Carbachol-evoked increase of intracellular calcium concentration in cerebellar granule cells was inhibited by ryanodine [30]. Activation of muscarinic acetylcholine receptors in NG108-15 neuroblastoma × glioma cells produces cyclic ADP-ribose, a known endogenous modulator of RyR, which may upregulate the release of calcium from the ryanodine receptors [18,19].…”

Type 1 and type 3 ryanodine receptors are selectively involved in muscarinic antinociception in mice: An antisense study

“…It should also be noted that an increased intracellular calcium mobilization through RyR activation has been demonstrated after stimulation of muscarinic receptors. Carbachol-evoked increase of intracellular calcium concentration in cerebellar granule cells was inhibited by ryanodine (Simpson et al, 1996). Activation of muscarinic acetylcholine receptors in NG108-15 neuroblastomaϫglioma cells produces cyclic ADP-ribose, a known endogenous modulator of RyR, which may upregulate the release of calcium from the RyRs (Higashida et al, 1997(Higashida et al, , 2001).…”