Investigating how calcium release from the endoplasmic reticulum (ER) is triggered and coordinated is crucial to our understanding of how oligodendrocyte progenitor cells (OPs) develop into myelinating cells. Sparks and puffs represent highly localized Ca 2ϩ release from the ER through ryanodine receptors (RyRs) and inositol trisphosphate receptors (IP 3 Rs), respectively. To study whether sparks or puffs trigger Ca 2ϩ waves in OPs, we performed rapid high-resolution line scan recordings in fluo-4-loaded OP processes. We found spontaneous and evoked sparks and puffs, and we have identified functional cross talk between IP 3 Rs and RyRs. Local events evoked using the IP 3 -linked agonist methacholine (MeCh) showed significantly different morphology compared with events evoked using the caffeine analog 3,7-dimethyl-1-propargylxanthine (DMPX). Pretreatment with MeCh potentiated DMPX-evoked events, whereas inhibition of RyRs potentiated events evoked by low concentrations of MeCh. Furthermore, activation of IP 3 Rs but not RyRs was critical for Ca 2ϩ wave initiation. Using immunocytochemistry, we show OPs express the specific Ca 2ϩ release channel subtypes RyR3 and IP 3 R2 in patches along OP processes. RyRs are coexpressed with IP 3 Rs in some patches, but IP 3 Rs are also found alone. This differential distribution pattern may underlie the differences in local and global Ca 2ϩ signals mediated by these two receptors. Thus, in OPs, interactions between IP 3 Rs and RyRs determine the spatial and temporal characteristics of calcium signaling, from microdomains to intracellular waves.

Glutamate is the primary excitatory transmitter in the suprachiasmatic nucleus (SCN). Ionotropic glutamate receptors (iGluRs) mediate transduction of light information from the retina to the SCN, an important circadian clock phase shifting pathway. Metabotropic glutamate receptors (mGluRs) may play a significant modulatory role. mGluR modulation of SCN responses to glutamate was investigated with fura-2 calcium imaging in SCN explant cultures. SCN neurons showed reproducible calcium responses to glutamate, kainate, and N-methyl-D-aspartate (NMDA). Although the type I/II mGluR agonists L-CCG-I and t-ACPD did not evoke calcium responses, they did inhibit kainate- and NMDA-evoked calcium rises. This interaction was insensitive to pertussis toxin. Protein kinase A (PKA) activation by 8-bromo-cAMP significantly reduced iGluR inhibition by mGluR agonists. The inhibitory effect of mGluRs was enhanced by activating protein kinase C (PKC) and significantly reduced in the presence of the PKC inhibitor H7. Previous reports show that L-type calcium channels can be modulated by PKC and PKA. In SCN cells, about one-half of the calcium rise evoked by kainate or NMDA was blocked by the L-type calcium channel antagonist nimodipine. Calcium rises evoked by K+ were used to test whether mGluR inhibition of iGluR calcium rises involved calcium channel modulation. These calcium rises were primarily attributable to activation of voltage-activated calcium channels. PKC activation inhibited K+-evoked calcium rises, but PKC inhibition did not affect L-CCG-I inhibition of these rises. In contrast, 8Br-cAMP had no effect alone but blocked L-CCG-I inhibition. Taken together, these results suggest that activation of mGluRs, likely type II, modulates glutamate-evoked calcium responses in SCN neurons. mGluR inhibition of iGluR calcium rises can be differentially influenced by PKC or PKA activation. Regulation of glutamate-mediated calcium influx could occur at L-type calcium channels, K+ channels, or at GluRs. It is proposed that mGluRs may be important regulators of glutamate responsivity in the circadian system.

Although metabotropic glutamate receptor (mGluR) modulation has been studied extensively in neurons, it has not been investigated in astrocytes. We studied modulation of glutamate-evoked calcium rises in primary astrocyte cultures using fura-2 ratiometric digital calcium imaging. Calcium plays a key role as a second messenger system in astrocytes, both in regulation of many subcellular processes and in long distance intercellular signaling. Suprachiasmatic nucleus (SCN) and cortical astrocytes showed striking differences in sensitivity to glutamate and to mGluR agonists, even after several weeks in culture. Kainate-evoked intracellular calcium rises were inhibited by concurrent application of the type I and II mGluR agonists quisqualate (10 M),. Inhibition mediated by L-CCG-I had longlasting effects (Ͼ45 min) in ϳ30% of the SCN astrocytes tested. The inhibition could be mimicked by the L-type calcium channel blocker nimodipine (1 M) as well as by protein kinase C (PKC) activators phorbol 12,13-dibutyrate (10 M) and phorbol 12-myristate 13-acetate (500 nM), and blocked by the PKC inactivator (Ϯ)-1-(5-isoquinolinesulfonyl)-2-methylpiperazine (200 M), suggesting a mechanism involving PKC modulation of L-type calcium channels. In contrast, mGluRs modulated serotonin (5HT)-evoked calcium rises through a different mechanism. The type III mGluR agonist L-2-amino-4-phosphonobutyrate consistently inhibited 5HT-evoked calcium rises, whereas in a smaller number of cells quisqualate and L-CCG-I showed both inhibitory and additive effects. Unlike the mGluR-kainate interaction, which required a pretreatment with an mGluR agonist and was insensitive to pertussis toxin (PTx), the mGluR modulation of 5HT actions was rapid and was blocked by PTx. These data suggest that glutamate, acting at several metabotropic receptors expressed by astrocytes, could modulate glial activity evoked by neurotransmitters and thereby influence the ongoing modulation of neurons by astrocytes.