We also show that activation of OXA-ir neurons in the medial TH occurs prior to the nocturnal onset of vigorous wheel-running. In this study, using c-Fos as a marker of cellular activation, we demonstrate that under constant light (LL), a condition that suppresses the amplitude of wheel-running rhythms, TH cells including OXA-immunoreactive (OXA-ir) neurons are much more active during the subjective night than the subjective day. Since orexin neuronal activation corresponds with forward locomotor activity, and because such behavior increases at night, it is unclear whether SCN-derived signals regulate the nighttime activation of orexin neurons. Indeed, orexin neurons demonstrate diurnal variation in activity/activation, with highest levels in nocturnal animals occurring during the night. Transgenic and pharmacological impairment of orexin-OXR1/R2 signaling implicates this neuropeptide system in the promotion of arousal and wakefulness. SCN efferents project to the TH and may directly contact orexin-containing cells. Immunohistochemical studies have established that these orexin-containing neurons have extensive neuronal projections and innervate key circadian structures including the SCN, IGL and MRN. In mammals, neurons expressing orexins are mostly limited to the lateral hypothalamic area and dorsomedial nucleus of the tuberal hypothalamus (TH). The orexins are mainly excitatory and mediate their biological actions through two G-protein coupled receptors, OXR1 and OXR2.

There are two bioactive forms, orexin-A (OXA) and orexin-B, which are cleaved from the common precursor prepro-orexin. Recently, significant progress was made in identifying the neurochemical basis of arousal with the isolation and characterization of the orexin/hypocretin neuropeptides (referred to as orexins in this study). Unfortunately, our understanding of how neural substrates integrate circadian and arousal information is far from complete. Photic information is conveyed to the SCN via the retinohypothalamic tract, while non-photic stimuli activate pathways originating in the thalamic intergeniculate leaflet (IGL) and the median raphe (MRN) of the brain stem. This SCN circadian clock is synchronized (entrained) by exogenous time cues (zeitgebers) such as varying levels of environmental light (photic stimuli), as well as by so-called non-photic cues that promote behavioral arousal. For example, within the suprachiasmatic nuclei (SCN), site of the brain's dominant circadian clock, neurons orchestrate daily rhythms responsible for a wide range of physiological and neural processes. The mammalian hypothalamus plays a fundamental role in the control of numerous critical brain and behavior states. Collectively these results demonstrate, for the first time, pronounced circadian control of orexin neuron activation and implicate recruitment of orexin cells in dark pulse resetting of the SCN circadian clock. In contrast, dark pulse exposure during the subjective night did not reset SCN-controlled behavioral rhythms and caused a transient suppression of neuronal activation in the TH. Concurrently, this stimulus suppressed SCN activity while activating cells in the median raphe. Moreover, exposure to a 6 h dark pulse during the subjective day, a stimulus that promotes arousal and phase advances behavioral rhythms, activated neurons in the medial and lateral TH including those containing orexin. Significantly, activation of orexin neurons in the medial and lateral TH occurred prior to the onset of sustained wheel-running activity. Here mice were placed in conditions of constant light, which suppressed locomotor activity, but also revealed a highly pronounced circadian pattern in orexin neuronal activation. Unfortunately, since orexin neurons are themselves activated by locomotor promoting cues, it is unclear how these two systems interact to regulate behavioral rhythms. Intriguingly, the SCN circadian clock can be reset by arousal-promoting stimuli while activation of orexin/hypocretin neurons is believed to be under circadian control, suggesting the existence of a reciprocal relationship. This interaction permits the daily rhythm of sleep and wake, regulated in parallel by circadian cues originating from the suprachiasmatic nuclei (SCN) and arousal-promoting signals arising from the orexin-containing neurons in the tuberal hypothalamus (TH). Temporal control of brain and behavioral states emerges as a consequence of the interaction between circadian and homeostatic neural circuits.

Dark pulse