How and why we sleep are central unsolved questions in medicine. Nearly 40 million people in the United States are estimated to experience chronic or intermittent sleep disorders such as narcolepsy, sleep apnea, restless leg syndrome and insomnia. Traditional approaches have identified several neuronal populations whose interplay is important in generating sleep and wakefulness. How that interplay is established, how it is altered in pathology and its cellular and molecular consequences, remain poorly understood. The long-term objective of this proposal is to determine the molecular identity and function of ion channels and receptors expressed by sleep-related neurons in order to understand the molecular mechanisms controlling sleep generation. Building on our findings from the previous funding period, this application focuses on the identity and function of voltage-gated calcium channels in controlling activity of mesopontine cholinergic neurons which are believed to play a pivotal role in the generation of wakefulness and REM sleep. Our central hypothesis is that the calcium influx through distinct calcium channels are differentially regulated by cholinergic and monoaminergic inputs and thereby play different roles in altering the integrative properties of mesopontine cholinergic neurons across behavioral state. To test this hypothesis we will use pharmacological methods with whole-cell patch clamp and single- and two-photon calcium imaging and laser uncaging methods in brain slices from wild-type, calcium channel knockout and muscarinic receptor knock-out mice. The results from these studies will 1) determine if Cav2.3 containing calcium channels are inhibited by cholinergic """"""""auto"""""""" receptors in the soma and dendrites of important arousal-related neurons in mouse; 2) determine which of the multiple muscarinc receptors expressed by these neurons inhibit these calcium channels. 3) determine which Ca2+ channels are inhibited by noradrenalin, serotonin and adenosine in the soma and dendrites of these neurons; 4) Determine the role of calcium influx through Cav2.3 channels in regulating dendritic and somatic excitability. These results will contribute to our understanding of the molecular basis of sleep regulation as well as continue to advance the mouse as a platform for future sleep research. ? ? ?
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