Christopher S. Leonard, Ph.D. - US grants

DESCRIPTION (provided by applicant): Chronic or intermittent sleep disorders such as narcolepsy, sleep apnea, and insomnia afflict nearly 40 million people in the United States. Yet the neural mechanisms controlling both normal sleep and its pathologies remain poorly understood. Considerable evidence indicates that mesopontine cholinergic neurons are critical for this control and that their disregulation is involved in narcolepsy, Parkinson's disease, supranuclear palsy and depression. The long term goal of this project is to understand the synaptic and non- synaptic mechanisms regulating activity of mesopontine cholinergic neurons. Recent compelling evidence indicates that disruption of the novel Hypocretin/Orexin (Hcrt/Orx) peptide system results in narcolepsy - a sleep disorder characterized by excessive daytime sleepiness, sleep fragmentation and the intrusion of rapid eye movement sleep behaviors into wakefulness. Building on the finding from the last funding period, which showed that mesopontine cholinergic neurons are important targets of these peptides, we will continue to investigate the general hypothesis that Hcrt/Orx peptides regulate both the short-term and long-term excitability of these neurons and that loss of Hcrt/Orx signaling upregulates the cholinergic phenotype of MPCh neurons and thereby contributes to the expression of cataplexy. To do so we will 1) continue to identify the ion channels activated by Hcrt/Orx and investigate their impact on the excitability of MPCh neurons;2) investigate the microsircuitry within the LOT to determine if functionaly related subsets of cholinergic neurons are differentially modulated by Hcrt/Orx and 3) determine if the upregulation of the enzymes responsible for cholinergic transmission following the loss of Hcrt/Orx signalling contributes to cataplexy. These experiments will use whole-cell patch clamp recording and calcium imaging methods in brain slices, tract tracing, tissue biochemistry, a novel extracellular probe to measure Ach release and behavioral pharmacology in control mice and mice lacking the two known orexin receptors to investigate these issues. Collectively, these results will advance the understanding of the molecular and cellular mechanisms underlying sleep regulation and its pathology.

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 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. This application focuses on the identity and function of a family of K+ channels subunit genes 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 K+ channels formed by Kv3 subunits regulate action potential shape, intracellular Ca2+ levels, repetitive firing and the release of transmitter from mesopontine cholinergic neurons. To test this hypothesis we will use pharmacological methods with whole-cell patch clamp recordings in brain slices from wild-type and Kv3 knock-out mice. The results of these studies will 1) identify and verify the intrinsic electrophysiological properties of important REM-sleep related neurons in mouse; 2) determine the molecular identity and function of native K+ channels formed by Kv3 subunits; 3) elucidate new mechanisms controlling the activity and release of transmitter by REM sleep-related neurons; 4) identify novel functions of Kv3 channels which have previously been associated with the fast-spiking phenotype rather than broad-spiking phenotype of brainstem cholinergic neurons. These results will contribute to our understanding of the molecular basis of sleep regulation as well as advancing the mouse as a platform for future sleep research.

DESCRIPTION (provided by applicant): 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.

DESCRIPTION (provided by applicant): Chronic or intermittent sleep disorders including narcolepsy, REM behavior disorder, sleep apnea, and insomnia afflict nearly 50-70 million people in the United States. Yet the neural mechanisms controlling both normal sleep and its pathologies remain poorly understood. Considerable evidence indicates that mesopontine cholinergic neurons and other neurons at the mesopontine junction are critical for this control and that their disregulation is involved in narcolepsy, REM behavior disorder, Parkinson's disease, supranuclear palsy and depression. The long term goal of this project is to understand the mechanisms regulating activity of these neurons and their functions in regulating sleep and sleep pathologies. Compelling evidence indicates that disruption of the Hypocretin/Orexin (Hcrt/Orx) peptide system results in narcolepsy - a sleep disorder characterized by excessive daytime sleepiness, sleep fragmentation and the intrusion of rapid eye movement sleep behaviors into wakefulness. Building on the findings from previous funding periods, which showed that mesopontine cholinergic neurons and dorsal raphe serotonergic neurons are important targets of these peptides, we will continue to investigate the general hypothesis that Hcrt/Orx peptides regulate both the short- term and long-term excitability of these neurons. We will explicitly test the hypothesis that cholinergic transmission resulting from mesopontine cholinergic neurons contribute the disruptions of waking and sleep in narcoleptic mice and that they promote the entrance into cataplexy but that their activity contributes little to regulating te duration of cataplectic attacks. To do so we will 1) identify the mechanisms underlying a new class of post-synaptic orexin actions post-spike afterhyperpolarization in mesopontine cholinergic neurons and dorsal raphe serotonergic neurons. 2) Explore the impact of the major orexin actions on firing pattern and encoding of inputs by mesopontine cholinergic neurons and dorsal raphe serotonergic neurons. 3) determine whether selective excitation and inhibition of mesopontine cholinergic neurons can regulate arousal and sleep in normal and narcoleptic mice and whether these neurons can trigger cataplexy in narcoleptic mice. These experiments will use whole-cell patch clamp recording, calcium imaging and dynamic clamp methods in brain slices from normal and orexin receptor knockout mice and will utilize optogenetic stimulation methods in normal and narcoleptic mice. Collectively, these results will advance our understanding of the molecular and cellular mechanisms underlying sleep regulation and its pathology.