Orexin A Promotes Wakefulness And Attenuates The Adenosine Induced Sleep In Rat Lateral Hypothalamus

The neuropeptide orexins, also called hypocretins, are produced by orexinergic neurons in the lateral hypothalamus area (LHA). Orexin system includes two separate peptides orexin A and B proteolytically derived from the same precursor protein and two specific G-protein-coupled receptors OX1R and OX2R.Although orexin neurons are localized in a subregion of hypothalamus, their projections and orexin receptors are widely distributed in the brain. The central orexin system plays a critical role in regulating sleep-wake cycle:the orexinergic neurons have extensive projections to the arousal systems; the loss of orexinergic cells or its malfunction has been linked to cataplexy like state in mice and abnormal rapid eye movement (REM) sleep. The orexin levels are low in cerebrospinal fluid and blood plasm. Orexins and orexin receptors play highly important roles in regulating sleep-awake states and the maintenance of arousal by regulating monoaminergic/cholinergic nuclei in the brain. The microinjection of orexin A into the lateral hypothalamus increase glutamate release, while glutamic acid stimulation of the lateral hypothalamic area promotes arousal and inhibits NREM/REM sleep. However, the mechanism of glutamatergic activation of orexin neurons on sleep-wake behavioral modulation is not known.Adenosine (AD), a byproduct of cell energy metabolism, serves as an effective sleep-promoting neurotransmitter in the central nervous system (CNS). Four subtypes of G protein-coupled adenosine receptors, A1R, A2aR, A2bR and A3R, are indicated in CNS, while A1Rs and A2aRs are involved in sleep-wake modulation. It is well illustrated that AD increases sleep by inhibition of the cholinergic region in basal forebrain through A1Rs. An in vitro study using electrophysiological methods reports that AD inhibits the activity of orexinergic neurons via A1Rs. A recent study in our lab has shown that AD inhibits the glutamatergic synaptic transmission to orexin neurons via A1Rs. Moreover, administration of adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) in the orexinergic LHA could induce sleep and blockade of A1R by application of a selective AD A1R antagonist 1,3-dipropyl-8-phenylxanthine(DPX) could produce a significant increase in wakefulness. These evidences illuminate that there is another important target involved the modulation of endogenous adenosine on sleep-wakefulness in the orexinergic zone of LHA. But until now, whether A2R is involves in the sleep-inducing effect of adenosine in the orexinergic LHA and whether the sleep-inducing effect of adenosine could be abolished by the orexins are still little known.In the present study, using polysomnogram (PSG) recordings and microinjection technique, we firstly observed this modulation of orexinergic neurons which involved in glutamatergic synaptic transmission on spontaneous sleep-wakefulness. Subsequently, we evaluate the inhibition of adenosine and adenosine receptors on the orexin neurons in the lateral hypothamus as well as the excitation of orexin on the sleep-inducing effect of adenosine. The results show as follow:1. The arousal effects of orexin AMicroinjection of 10 pmol orexin A into LHA could increase the total amount of wakefulness to 1.28 and 1.31-fold and decrease the entire time of NREM sleep to 0.90 and 0.89-fold during the first 3-h period, when compared with that of the ACSF treatment and baseline respectively. The change of REM sleep was under statistical level. Similarly, application of 40pmol orexin A into LHA significantly increased the total amount of wakefulness to 1.32 and 1.35-fold during the first 3-h session compared with the ACSF and baseline day respectively. The increase in wakefulness induced by 40 pmol orexin A was concomitant with the reduction in NREM/REM sleep。The time of wakefulness was increased after DMSO/orexin A application during the first 3-h post-injection sessions compared with control, and then recovered approximately to control level after SB-334867/orexin-A treatment.2. Orexin A promotes wakefulness via local glutamatergic neuronsThe significant increase in wakefulness to 1.39-fold (P<0.05) was concomitant with the decrease in NREM sleep to 79%(P<0.05) after the high dose of 80 ng glutamate microinjection during the 3-h post-injection recording period, when compared with ACSF treatment group. There is no significant change in any behavioral state after 40 ng glutamate administation. The ACSF/orexin A administration significantly increased the total amount of wakefulness during the 3-h sessions to 1.36-fold when compared with control, and then reduced approximately to control level after NMD A antagonist D-AP5/orexin A treatment.3. The sleep-inducing effect of ADAD administration to LHA at doses of 1,10,20 nmol decreased the total amount of wakefulness to 84%,62% and 60% during that first 3-h recording period and concomitantly increased NREM and REM sleep, when compared with baseline. AD administration into the LHA at 20 nmol dose shortened the wake time to 63%,61%,53% and 78% at the 1-h, 2-h,3-h and 5-h, respectively, over the ACSF control group. The reduction of wakefulness was concomitant with enhancements in NREM and REM. Microinjection of a selective AD A1R antagonist DPX into LHA could increase wakefulness and decrease sleep, but there is on similar effect on A2R antagonist DMPX application, suggesting A1R but not A2R involves in the sleep-inducing effect of AD.4. Orexin A attenuates the sleep-inducing effect of ADThe total amount of wakefulness after AD/ACSF microinjection was significantly decreased to 74%(P<0.05) during the first 3-h post-injection sessions compared with the baseline, and then recovered approximately to baseline level after AD/orexin A treatment. Accordingly, the NREM and REM sleep was respectively increased to 1.10 and 1.40-fold by microinjection of AD, when compared with baseline, and then both recovered approximately to baseline level after AD/orexin A treatment. Moreover, by comparing the sleep-wake regulation of AD/ACSF and AD/orexin A, we find orexin A inhibits the sleep-inducing effect of AD.In conclusion, these observations provide evidences that the orexin A could promote wake and concomitantly decrease NREM and REM sleep via local glutamate circuits. In addition, AD reduces the amount of wakefulness through A1Rs, but not A2Rs. Furthermore, the sleep-inducing effect of adenosine on sleep-wakefulness is attenuated when orexin A tone was increased.