Nitric oxide synthase (NOS)-containing mesopontine cholinergic (MPCh) neurons of the laterodorsal tegmental nucleus (LDT) are hypothesized to drive the behavioral states of waking and REM sleep through a tonic increase in firing rate which begins before and is maintained throughout these states. In principle, increased firing could elevate intracellular calcium levels and regulate numerous cellular processes including excitability, gene expression, and the activity of neuronal NOS in a state-dependent manner. We investigated whether repetitive firing, evoked by current injection and N-methyl-D-aspartate (NMDA) receptor activation, produces somatic and proximal dendritic [Ca2+](i) transients and whether these transients are modulated by serotonin, a transmitter thought to play a critical role in regulating the state-dependent firing of MPCh neurons. [Ca2+](i) was monitored optically from neurons filled with Ca2+ indicators in guinea pig brain slices while measuring membrane potential with sharp microelectrodes or patch pipettes. Neither hyperpolarizing current steps nor subthreshold depolarizing steps altered [Ca2+](i). In contrast, suprathreshold currents caused large and rapid increases in [Ca2+](i) that were related to firing rate. TTX (1 μM) strongly attenuated this relation. Addition of tetraethylammonium (TEA, 20 mM), which resulted in Ca2+ spiking on depolarization, restored the change in [Ca2+](i) to pre-TTX levels. Suprathreshold doses of NMDA also produced increases in [Ca2+](i) that were reduced by up to 60% by TTX. Application of 5-HT, which hyperpolarized LDT neurons without detectable changes in [Ca2+](i), suppressed both current- and NMDA-evoked increases in [Ca2+](i) by reducing the number of evoked spikes and by inhibiting spike-evoked Ca2+ transients by ~40% in the soma and proximal dendrites. This inhibition was accompanied by a subtle increase in the spike repolarization rate and a decrease in spike width, as expected for inhibition of high-threshold Ca2+ currents in these neurons. NADPH-diaphorase histochemistry confirmed that recorded cells were NOS-containing. These findings indicate the prime role of action potentials in elevating [Ca2+](i) in NOS-containing MPCh neurons. Moreover, they demonstrate that serotonin can inhibit somatic and proximal dendritic [Ca2+](i) increases both indirectly by reducing firing rate and directly by decreasing the spike-evoked transients. Functionally, these data suggest that spike-evoked Ca2+ signals in MPCh neurons should be largest during REM sleep when serotonin inputs are expected to be lowest even if equivalent firing rates are reached during waking. Such Ca2+ signals may function to trigger Ca2+-dependent processes including cfos expression and nitric oxide production in a REM-specific manner.
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