Characterization of the metabotropic glutamate receptors mediating phospholipase C activation and calcium release in cerebellar granule cells: calcium‐dependence of the phospholipase C response

Abstract

In this study we have determined the metabotropic glutamate receptors (mGluRs) involved in the glutamate activation of phospholipase C (PLC) and Ca(2+) mobilization in cerebellar granule cells at 9 days in vitro; and studied the Ca(2+) modulation of the PLC response. Both PLC activation and Ca(2+) signalling were found to be mediated exclusively by the mGluR1 subtype, although both group I mGluRs, mGluR1 alpha and mGluR5, could be detected in cell extracts. Exposure of cells to medium devoid of Ca(2+) for various times before agonist stimulation reduced the PLC response, which was quickly recovered following the re-exposure of cells to Ca(2+)-containing medium. The extent of the glutamate response correlated well with changes in the cytosolic Ca(2+) concentration. On the other hand, loading of the intracellular Ca(2+) stores by a transient depolarization followed by washing in nondepolarizing buffer, allowed glutamate to release stored Ca(2+) in the majority of cells and enhanced glutamate activation of PLC. Under such conditions, the absence of extracellular Ca(2+) during stimulation and the chelation of cytosolic Ca(2+) with BAPTA/AM inhibited both glutamate-elicited Ca(2+) response and PLC activation. Overall, these results indicate that the mGluR-mediated activation of PLC depends on the presence of extracellular Ca(2+) and can be modulated by moderate changes of cytosolic Ca(2+). Furthermore, ryanodine reduced PLC stimulation by glutamate in predepolarized cells but not in control cells, suggesting that ryanodine receptors could play a role in the potentiation of the mGluR-mediated activation of PLC by Ca(2+) release in predepolarized cells.

Costimulation of AMPA and Metabotropic Glutamate Receptors Underlies Phospholipase C Activation by Glutamate in Hippocampus

“…Thus, the lack of PH␦-GFP translocation by DHPG does not mean that DHPG has no effect, but that PH␦-GFP translocation assay is not sensitive enough to detect small changes. In fact, previous results ob-tained from the IP formation assay do not contradict but rather agree with our results: DHPG and glutamate increased 3 H-InsPs accumulation to 362 and 1344% of the basal level, respectively (Masgrau et al, 2001). The merit of PH␦-GFP translocation assay is that we can monitor dynamic changes at the single-cell level to investigate underlying mechanisms of PLC activation.…”

“…Our data provide novel insight into the interaction between AMPA receptors and group I mGluRs as well as the Ca 2ϩ dependence of mGluR5-PLC signaling cascades in hippocampal neurons. Ca 2ϩ dependence of PLC activation by group I mGluRs and muscarinic receptors was previously reported (Masgrau et al, 2000(Masgrau et al, , 2001Hashimotodani et al, 2005;Maejima et al, 2005). In cerebellar granule cells, activation of PLC by glutamate, which is mediated by mGluR1, was dependent on the external Ca 2ϩ (Masgrau et al, 2001), whereas muscarinic receptor-mediated activation of PLC was modulated by changes in the loading state of intracellular Ca 2ϩ stores (Masgrau et al, 2000).…”

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

“…Local Ca 2ϩ control by synaptic stimuli is commonly observed in spines and smooth dendrites of pyramidal neurons (Svoboda et al, 1996;Sabatini et al, 2001Sabatini et al, , 2002Augustine et al, 2003;Goldberg et al, 2003). Ca 2ϩ signals observed in the soma of GrCs in culture (Irving et al, 1992a,b;del Rio et al, 1999;Masgrau et al, 2001) may reflect different recording or cellular conditions, as well as the massive, protracted nonlocalized stimulation caused by bath perfusion of high-K ϩ solutions or glutamate receptor agonists.…”

“…Glutamate released by mf discharge activated NMDA-Rs and mGlu-Rs (D' Angelo et al, 1993Angelo et al, , 1995Rossi et al, 1996). NMDA-Rs are permeable to Ca 2ϩ , and mGlu-Rs probably act through the IP 3 pathway, which is effectively stimulated by glutamate in GrCs (del Rio et al, 1999;Masgrau et al, 2001;Monti et al, 2002). During repetitive synaptic transmission, the main contribution to [Ca 2ϩ ] i was given by NMDA-R, whereas mGlu-R1 could play an amplifying role (Finch et al, 1991;Irving et al, 1992b), explaining the need for NMDA-R and mGlu-R coactivation in mf-GrC LTP induction (Rossi et al, 1996;D'Angelo et al, 1999;Maffei et al, 2002).…”

“…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.…”