Abstract
Orexin/hypocretin neurons play a crucial role in the regulation of sleep/wakefulness, primarily in the maintenance of wakefulness. These neurons innervate wide areas of the brain and receive diverse synaptic inputs including those from serotonergic (5-HT) neurons in the raphe nucleus. Previously we showed that pharmacological application of 5-HT directly inhibited orexin neurons via 5-HT1A receptors. However, it was still unclear how 5-HT neurons regulated orexin neurons since 5-HT neurons contain not only 5-HT but also other neurotransmitters. To reveal this, we generated new triple transgenic mice in which orexin neurons express enhanced green fluorescent protein (EGFP) and 5-HT neurons express channelrhodopsin2 (ChR2). Immunohistochemical studies show that nerve endings of ChR2-expressing 5-HT neurons are in close apposition to EGFP-expressing orexin neurons in the lateral hypothalamic area. Using these mice, we could optogenetically activate 5-HT nerve terminals and record postsynaptic effects from orexin neurons. Activation of nerve terminals of 5-HT neurons directly inhibited orexin neurons via the 5HT1A receptor, and also indirectly inhibited orexin neurons by facilitating GABAergic inhibitory inputs without affecting glutamatergic inputs. Increased GABAergic inhibitory inputs in orexin neurons were confirmed by the pharmacological application of 5-HT. These results suggest that orexin neurons are inhibited by 5-HT neurons, primarily via 5-HT, in both direct and indirect manners.
Highlights
Orexin/hypocretin neurons play a crucial role in the regulation of sleep/wakefulness, primarily in the maintenance of wakefulness
We generated triple transgenic orexin-enhanced green fluorescent protein (EGFP); Tph2-tTA; TetO ChR2 mice (Fig. 1a). These triplegenic mice expressed EGFP in orexin neurons under control of the human prepro-orexin promoter[27], and a tetracycline-controlled transactivator exclusively in 5-HT neurons in the raphe nucleus under the control of the Tph[2] promoter. tTA binds to the tetracycline operator (TetO) sequence and induces ChR2 expression[28]
Orexin had initially been identified as an important neuropeptide that regulates feeding behaviour[1], the peptide was subsequently found to be crucial in maintaining wakefulness[35]
Summary
Orexin/hypocretin neurons play a crucial role in the regulation of sleep/wakefulness, primarily in the maintenance of wakefulness These neurons innervate wide areas of the brain and receive diverse synaptic inputs including those from serotonergic (5-HT) neurons in the raphe nucleus. Orexin functions as a neuropeptide that binds to G-protein coupled receptors (GPCRs), termed orexin 1 (OX1R) and orexin 2 (OX2R) receptors, and participates in multiple physiological responses including feeding behaviour, sleep/wakefulness, and perception of pain[1,4,5,6] To regulate such intricate physiological responses, orexin neurons receive and integrate diverse synaptic inputs from many brain regions, including serotonergic neurons in the midbrain raphe nuclei[7]. The comprehensive effect of 5-HTergic regulation of orexin neurons could be complex and is largely unknown
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