The Hypothalamus provides the heaviest innervation of the orexin neurons, with the densest inputs originating in the POA and PH. Most of hypothalamic regions innervate the medial part of the orexin field. Inputs to orexin neurons from the hypothalamic area are:

These afferents strongly suggest that orexin neurons are associated with many hypothalamic functions. In addition to above inputs, orexin neurons receive local excitatory input between themselves and/or from glutamatergic interneurons (Li et al. 2002; Sakurai et al. 2005; Yamanaka et al. 2010). This self-conditioning mechanism may help orexin neurons remain active for a long time when necessary, for example, to arouse animals during a variety of active behavior (España et al. 2003; Mochizuki et al. 2004, 2006; Alexandre et al. 2013). In contrast, the heaviest, presumably inhibitory input originates in the VLPO and MPA; possible sleep-active neurons that produce GABA and galanin (Sherin et al. 1998; McGinty et al. 2004). The VLPO mainly projects to the medial and perifornical parts of the orexin field, and orexin neurons in these regions show the greatest reduction in the activity during sleep (Estabrooke et al. 2001). Regions that control metabolism and feeding such as the Arc, DMH and VMH are likely to modulate the orexin neurons.

Sleep/wake behavior is closely regulated by the circadian biological rhythm, however, orexin neurons rarely receive the direct projection from the SCN, the main circadian oscillator in the mammalian brain (Reppert and Weaver 2002). An alternative multi-synaptic pathway was suggested by the neuroanatomical tracing and behavioral studies that the output of the SCN was first mediated by the neurons in the subparaventricular zone (SPZ), then to the DMH and finally to orexin neurons (Lu et al. 2001; Chou et al. 2003; Saper et al. 2005, 2010).

Most inputs from the forebrain regions innervate orexin neurons diffusely across the entire field, with slightly more projections in the perifornical region. Inputs to orexin neurons from the forebrain and limbic areas are:

These forebrain inputs may drive orexin neurons in response to various emotional, stress and autonomic stimuli. The prefrontal cortex has an important role in emotional control, and suppression of these areas reduced food-elicited cataplexy in orexin knockout mice (Oishi et al. 2013). The amygdala is also one of the well-studied regions for processing emotional stimuli, and large neurotoxic lesion in the CeM and basolateral amygdala reduced emotion/motivation-triggered cataplexy in orexin knockout mice (Burgess et al. 2013). The cholinergic neurons in the basal forebrain, widely spread in the septal nuclei, the diagonal band of Broca, the magnocellular preoptic nucleus (MCPO) and the SI, are supposed to work for wake maintenance, and 20 % of orexin neurons were excited electrophysiologycally with a muscarinic agonist, carbachol (Yamanaka et al. 2003; Sakurai et al. 2005). However, more recent study suggested that the major afferents from the basal forebrain are non-cholinergic innervations (Agostinelli et al. 2013). Other inputs, such as the AcbSh and BST would influence stress, aggression, and anxiety, and all of these forebrain inputs may be important to activate orexin neurons when emotional stimuli drive arousal and autonomic responses in animals (Kayaba et al. 2003).

Projections from the SNR, DR, and VTA preferentially innervate orexin neurons in the lateral part of the orexin field. Many of these brainstem inputs project primarily to the ipsilateral orexin field, but fibers from the DR, ventrolateral PAG, and LC project bilaterally. Inputs to orexin neurons from brainstems are:

Compared to the dense fiber projections from orexin neurons to wake-active nuclei in the brain stem (LC, DR, PB), afferent signals from these groups were considered minor. These neurons may work mostly as output pathway of orexin signaling. Many serotonergic neurons around the MnR innervate orexin neurons and may form a feedback circuit to modulate the activity of orexin neurons (Muraki et al. 2004). Orexin neurons in the lateral part of the field are activated when an animal anticipates a reward such as drugs or food (Harris et al. 2005), and these neurons may be more influenced by regions that regulate reward, emotion, and autonomic function such as the VTA, SN, or ascending visceral afferents.

Loss of orexin signals in the brain causes narcolepsy syndrome in both humans and animals, showing chronic sleepiness, fragmented sleep/wake and cataplexy. One way to map the critical brain regions for orexin neurotransmission is to focally restore the orexin signaling and improve the behavioral deficits in narcoleptic animals. For this purpose, we produced a new line of mouse model in which a loxP-flanked gene-targeting cassette disrupts the transcription of OX2 receptor gene, but normal, eutopic expression of OX2 receptor can be restored by introducing Cre recombinase in the neurons (Mochizuki et al. 2011). These mice were born as OX2 receptor null mice, and had mildly fragmented wakefulness during the active period. We focally injected an adeno-associated viral vector (AAV) coding for Cre recombinase into the posterior hypothalamus, including the TMN (histamine neurons) and the SuM (probably glutamate neurons), and found that the restoration of OX2 in these neurons completely rescued the fragmented wakefulness in these mice. The results indicated that the posterior hypothalamic region works as an important relay pathway of orexin signaling to consolidate wake behavior. However, similar OX2 receptor rescue using OX2-coding viral vector was examined in constitutive OX1/OX2 double receptor knockout mice, showing severe sleep/wake fragmentation than the single receptor mutants, and the induction of OX2 receptor in the posterior hypothalamus was not effective to improve the fragmented wake (Hasegawa et al. 2014). The results suggest that arousal signal from the posterior hypothalamus may need further OX1-mediated downstream relays to fully recover the wake deficit in narcoleptic mice.