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Neuronal nitric oxide synthase (nNOS) is broadly expressed in the brain and associated with synaptic plasticity through NMDAR-mediated calcium influx. However, its physiological activation and the mechanisms by which nitric oxide (NO) influences synaptic transmission have proved elusive. Here, we exploit the unique input-specificity of the calyx of Held to characterize NO modulation at this glutamatergic synapse in the auditory pathway. NO is generated in an activity-dependent manner by MNTB principal neurons receiving a calyceal synaptic input. It acts in the target neuron and adjacent inactive neurons to modulate excitability and synaptic efficacy, inhibiting postsynaptic Kv3 potassium currents (via phosphorylation), reducing EPSCs and so increasing action potential duration and reducing transmission fidelity. We conclude that NO serves as a volume transmitter and slow dynamic modulator, integrating spontaneous and evoked neuronal firing, thereby providing an index of global activity and regulating information transmission across a population of active and inactive neurons.

In this study we have quantitatively assessed the basal turnover of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) and M 3 -muscarinic receptor-mediated changes in phosphoinositides in the human neuroblastoma cell line, SH-SY5Y. We demonstrate that the polyphosphoinositides represent a minor fraction of the total cellular phosphoinositide pool and that in addition to rapid, sustained increases in [ 3 H]inositol phosphates dependent upon the extent of receptor activation by carbachol, there are equally rapid and sustained reductions in the levels of polyphosphoinositides. Compared with phosphatidylinositol 4-phosphate (PtdIns(4)P), PtdIns(4,5)P 2 was reduced with less potency by carbachol and recovered faster following agonist removal suggesting protection of PtdIns(4,5)P 2 at the expense of PtdIns(4)P and indicating specific regulatory mechanism(s). This does not involve a pertussis toxin-sensitive G-protein regulation of PtdIns(4)P 5-kinase. Using wortmannin to inhibit PtdIns 4-kinase activity, we demonstrate that the immediate consequence of blocking the supply of PtdIns(4)P (and therefore PtdIns(4,5)P 2 ) is a failure of agonist-mediated phosphoinositide and Ca 2؉ signaling. The use of wortmannin also indicated that PtdIns is not a substrate for receptor-activated phospholipase C and that 15% of the basal level of PtdIns(4,5)P 2 is in an agonist-insensitive pool. We estimate that the agonist-sensitive pool of PtdIns(4,5)P 2 turns over every 5 s (0.23 fmol/cell/min) during sustained receptor activation by a maximally effective concentration of carbachol. Immediately following agonist addition, PtdIns(4,5)P 2 is consumed >3 times faster (0.76 fmol/cell/min) than during sustained receptor activation which represents, therefore, utilization by a partially desensitized receptor. These data indicate that resynthesis of PtdIns(4,5)P 2 is required to allow full early and sustained phases of receptor signaling. Despite the critical dependence of phosphoinositide and Ca 2؉ signaling on PtdIns(4,5)P 2 resynthesis, we find no evidence that this rate resynthesis is limiting for agonist-mediated responses.

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2؉oscillations arising through activation of the metabotropic glutamate receptor mGluR5a expressed in Chinese hamster ovary cells and find that these oscillations are largely insensitive to agonist concentration. Using an inducible receptor expression system and a non-competitive antagonist, in conjunction with the translocation of eGFP-PH PLC␦ to monitor inositol 1,4,5-trisphosphate (InsP 3 ) oscillations in single cells, we show that mGluR5a density determines the frequency of these oscillations. The predominant underlying mechanism resulted from a negative feedback loop whereby protein kinase C (PKC) inhibited InsP 3 generation. Down-regulation of PKC by prolonged exposure to phorbol ester revealed a second form of Ca

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