Emotions, stress, hunger, and circadian rhythms all promote wakefulness and behavioral arousal. Little is known about the pathways mediating these influences, but the orexin-producing neurons of the hypothalamus may play an essential role. These cells heavily innervate many wake-promoting brain regions, and mice lacking the orexin neurons have narcolepsy and fail to rouse in response to hunger (Yamanaka et al. [2003] Neuron 38:701-713). To identify the afferents to the orexin neurons, we first injected a retrograde tracer into the orexin neuron field of rats. Retrogradely labeled neurons were abundant in the allocortex, claustrum, lateral septum, bed nucleus of the stria terminalis, and in many hypothalamic regions including the preoptic area, dorsomedial nucleus, lateral hypothalamus, and posterior hypothalamus. Retrograde labeling in the brainstem was generally more modest, but labeling was strong in the periaqueductal gray matter, dorsal raphe nucleus, and lateral parabrachial nucleus. Injection of an anterograde tracer confirmed that most of these regions directly innervate the orexin neurons, with some of the heaviest input coming from the lateral septum, preoptic area, and posterior hypothalamus. In addition, hypothalamic regions preferentially innervate orexin neurons in the medial and perifornical parts of the field, but most projections from the brainstem target the lateral part of the field. Inputs from the suprachiasmatic nucleus are mainly relayed via the subparaventricular zone and dorsomedial nucleus. These observations suggest that the orexin neurons may integrate a variety of interoceptive and homeostatic signals to increase behavioral arousal in response to hunger, stress, circadian signals, and autonomic challenges.

Sleep is one of the few major whole-organ phenomena for which no function and no underlying mechanism have been conclusively demonstrated. Sleep could result from global changes in the brain during wakefulness or it could be regulated by specific loci that recruit the rest of the brain into the electrical and metabolic states characteristic of sleep. Here we address this issue by exploiting the genetic tractability of the fruitfly, Drosophila melanogaster, which exhibits the hallmarks of vertebrate sleep. We show that large changes in sleep are achieved by spatial and temporal enhancement of cyclic-AMP-dependent protein kinase (PKA) activity specifically in the adult mushroom bodies of Drosophila. Other manipulations of the mushroom bodies, such as electrical silencing, increasing excitation or ablation, also alter sleep. These results link sleep regulation to an anatomical locus known to be involved in learning and memory.

Narcolepsy is caused by a lack of orexin (hypocretin), but the physiologic process that underlies the sleepiness of narcolepsy is unknown. Using orexin knock-out (KO) mice as a model of narcolepsy, we critically tested the three leading hypotheses: poor circadian control of sleep and wakefulness, inadequate activation of arousal regions, or abnormal sleep homeostasis. Compared with wild-type (WT) littermates, orexin KO mice had essentially normal amounts of sleep and wake, but wake and non-rapid eye movement (NREM) bouts were very brief, with many more transitions between all behavioral states. In constant darkness, orexin KO mice had normal amplitude and timing of sleep-wake rhythms, providing no evidence for disordered circadian control. When placed in a new, clean cage, both groups of mice remained awake for ϳ45 min, demonstrating that, even in the absence of orexin, fundamental arousal regions can be engaged to produce sustained wakefulness. After depriving mice of sleep for 2-8 hr, orexin KO mice recovered their NREM and rapid eye movement sleep deficits at comparable rates and to the same extent as WT mice, with similar increases in EEG delta power, suggesting that their homeostatic control of sleep is normal. These experiments demonstrate that the fragmented wakefulness of orexin deficiency is not a consequence of abnormal sleep homeostasis, poor circadian control, or defective fundamental arousal systems. Instead, the fragmented behavior of orexin KO mice may be best described as behavioral state instability, with apparently low thresholds to transition between states.

Summary An understanding of sleep requires the identification of distinct cellular circuits that mediate the action of specific sleep:wake-regulating molecules, but such analysis has been very limited. We identify here a circuit that underlies the wake-promoting effects of octopamine in Drosophila. Using MARCM, we identified the ASM cells in the medial protocerebrum as the wake-promoting octopaminergic cells. We then blocked octopamine signaling in random areas of the fly brain and mapped the post-synaptic effect to insulin-secreting neurons of the pars intercerebralis (PI). These PI neurons show altered potassium channel function as well as an increase in cAMP in response to octopamine, and genetic manipulation of their electrical excitability alters sleep:wake behavior. Effects of octopamine on sleep:wake are mediated by the cAMP-dependent isoform of the OAMB receptor. These studies define the cellular and molecular basis of octopamine action and suggest that the PI is a sleep:wake-regulating neuroendocrine structure like the mammalian hypothalamus.

Narcolepsy with cataplexy is likely caused by a loss of the orexin-producing neurons. In addition, loss of dynorphin and neuronal activity-regulated pentraxin may contribute to the symptoms of narcolepsy.