Orexins/Hypocretins are newly discovered hypothalamic peptides that studies in canine inherited narcolepsy and mouse knock-out experiments link to cataplexy/narcolepsy, which may be regarded as disorders of REM sleep and wakefulness. Indeed, orexin neurons in the lateral hypothalamus have widespread projections to regions important in control of sleep and wakefulness. The broad objective of this program of research is to understand the physiological and pharmacological mechanisms by which orexin modulates behavioral state and thereby to provide a sound basis for the understanding and treatment of human sleep disorders, especially narcolepsy. The key techniques to be used are novel combinations of multi-disciplinary methods including the use of microdialysis perfusion of antisense against mRNA of orexin receptors combined with electrographic recording of behavioral states, measurement of extracellular levels of orexin peptides using enzyme linked immunosorbent assay (ELISA), and a method combining extracellular single unit recording with microdialysis-delivered orexinA and B in freely behaving rats. Our broad hypotheses are that (1)orexin/hypocretin controls/regulates REM sleep and cataplexy via selective action on brainstem neurons, primarily those in the nucleus sub-coeruleus alpha, and that this action is mediated by the orexin II receptor; and (2)that orexin/hypocretin regulates wakefulness through the forebrain sites of the cholinergic basal forebrain and tubero-mammillary nucleus (TMN), with mediation by the orexin II receptor. Microdialysis perfusion of antisense against the mRNA of orexin receptors will be used to reduce the levels of orexin receptors, producing a """"""""reversible knockout."""""""" We will test the hypothesis that, in accord with our preliminary data, orexin-B has a major effect on REM-related phenomena via orexin type II receptors in the subcoeruleus region of the pontine reticular formation, and that antisense to this receptor will increase muscle atonia and the REM phase of sleep, as well as cataplexy. We hypothesize that orexin acts via type I receptors in the locus coeruleus and dorsal raphe, predicting that antisense to type I receptors will increase REM sleep but not cataplexy. Conversely, we predict that microdialysis application of orexin B and A (relatively selective for type I receptors) respectively to these regions will decrease REM sleep. Microdialysis applications of antisense and orexin-B in brainstem muscle inhibitory pathways will test for the presence of predicted orexin modulatory effects on cataplexy. For mesopontine cholinergic neurons, we predict that orexin-B mediates arousal via activating neurons preferentially active in both wakefulness and REM sleep, and this will be tested via microdialysis-applied orexin-B combined with unit recording. In the forebrain, we hypothesize that orexin promotes wakefulness by excitatory actions on basal forebrain and histaminergic neurons preferentially active in this state, and modulated by orexin II receptors, a hypothesis supported by our preliminary data on basal forebrain microdialysis perfusion of orexin peptide. In experiments whose logic parallels those in the brainstem (but with wakefulness as the primary variable), we will use perfusion of antisense, orexin-B and electrographic and unit recording to test this hypothesis. Throughout all brainstem and forebrain sites, we predict that the extracellular levels of orexin-A will be highest during the dark (active) phase compared with the light phase and orexin-B will be highest in wakefulness as compared to non-REM phases of sleep.
