Effect Of Adenosine And Its Receptor On Sleep Awakening In Ventrolateral Medial Area And Its Mechanism

Sleep is regulated by circadian rhythm and homeostasis. Adenosine is a critical endogenous sleep-promoting substance, which is involved in the regulation of sleep. There are four subtypes of adenosine receptors (R), including A1R, A2AR, A2BR and A3R. Among them, A1R and/or A2AR subtypes have been reported to mediate the sleep-promoting effect of adenosine. Several lines of evidence suggest that adenosine acts via A1R at the wake-promoting areas of cholinergic basal forebrain, histaminergic tuberomammillary nucleus, and/or orexinergic lateral hypothalamus to promote sleep. However, systemical or intracerebroventricular administration of an A1R agonist has no clear effects on sleep promotion. In addition, A1R knockout mice do not show any phenotypes on sleep under baseline conditions or after sleep deprivation. Thus, we hypothesized that the effect of adenosine is site-dependent and activation of A1R in some brain regions may inhibit sleep. The ventrolateral preoptic nucleus (VLPO) has been considered to be a critical region for sleep induction. We hypothesized that activation of A1R may inhibit the sleep active neurons in the VLPO, and thus promote wakefulness, counteracting the effect caused by inhibition of wake-promoting systems. In this study, we investigated the role of A1R in the VLPO on the regulation of spontaneous and homeostatic sleep by employing a highly automatic recording and analysis system for sleep-wake profile, pharmacology, immunohistochemistry, and A1R knockout mice.Methods1. We employed electroencephalogram (EEG) and electromyogram (EMG) to evaluate the effect of adenosine, A1R agonist (N6-cyclopentyladenosine, CPA), antagonist (1,3-dimethyl-8-cyclopenthylxanthine, CPT), A2AR agonist2-(4-(2-carboxyethyl)phenylethylamino)-adenosine-5’-N-ethylcarboxamideadeno sine, CGS21680) or antagonist ((E)-phosphoric acid mono-[3-[8-[2-(3-metho-xyphenyl) vinyl]-7-methyl-2,6-dioxo-l-prop-2-ynyl-1,2,6,7-tetrahydropurin-3-yl]propyl] ester), MSX-3) administered into rat or mouse VLPO on sleep-wake profile.2. We employed the sleep deprivation paradigm combined with EEG recording and microinjection method to study the role of A1R in the VLPO in the regulation of sleep homeostasis.3. We used an immunohistochemcal staining method to detect Fos protein in different wake-related areas including locus coeruleus (LC), ventrolateral periaqueductal gray (VLPAG), tuberomammillary nucleus (TMN), dorsal raphe nucleus (DRN) and substantia nigra (SN) after CPA admisnistraion into the rat VLPO.4. We engaged the immunohistochemical staining method to detect the expression of A1R in the VLPO under baseline condition or after sleep deprivation, respectively.ResultsThe effect of A1R and A2AR in the VLPO on spontaneous sleep-wake profiles and the mechanism involvedA1R agonist, CPA, given at0.17,0.5or1.5nmol/side bilaterally significantly decreased the total amount of NREM and REM sleep in a dose-dependent manner during2h after the injection into rat VLPO, with a concomitant increase of wakefulness in comparison with the vehicle injection. These results clearly indicate that activation of A1R in the VLPO promotes wakefulness. Meanwhile, we found that CPA induced complete wakefulness immediately after the injection, indicating that A1R in the VLPO is involved in the induction of wakefulness.CPT given at6,18.5or55nmol/side significantly increased the total amount of NREM sleep during5h after the injection into rat VLPO, with a concomitant decrease of wakefulness in comparison with the vehicle injection. REM sleep had the tendency to increase, but not statistically different. To better understand the profile of NREM sleep behavior after CPT administration at a dose of55pmol/side, we determined the bout distribution of three stages as a function of bout duration. After CPT administration, short NREM sleep bouts (<64s) and wake bouts (<16s), namely, brief awakenings, increased, while long NREM sleep and wake bouts remained unchanged, resulting in increased numbers of total NREM sleep and wake episodes and shortened duration of wake episodes. Furthermore, the number of state transitions from NREM to wakefulness, from wakefulness to NREM and from REM sleep to wakefulness increased. In addition, the frequency range of0.75-3.25Hz of delta wave increased. EEG power distribution during REM sleep and wakefulness was unchanged. These results clearly showed that blockade of A1R in the VLPO increased the quantity and quality of NREM sleep, indicating the physiological role of A1R in the VLPO on the regulation of sleep and wakefulness. Increased brief awakenings and fragmentation of increased NREM sleep after CPT treatment indicated the difficulties for animals to maintain wakefulness and that A1R in the VLPO play a important role in maintaining wakefulness.CPA at0.3nmol/side decreased NREM and REM sleep for1h in WT mice after the injection into the VLPO as compared with that of the baseline day. However, A1R KO mice did not display any significant changes in NREM and REM sleep after the CPA administration, indicating that A1R is crucial for the increased wakefulness after CPA injection into the VLPO.A2AR agonist, CGS21680, given at6,55or500pmol/side significantly increased the total amount of NREM and REM sleep in a dose-dependent manner during5h after the injection into rat VLPO, with a concomitant decrease of wakefulness in comparison with the vehicle injection. After CGS21680treatment at500pmol/side, total NREM and REM sleep episode numbers increased and mean duration of wakefulness decreased. There was an increase in the number of different lengths of NREM and REM sleep, which caused decreased long wake bouts and increased short wake bouts. No changes were observed between transitions of each stage except for the transition from NREM to REM sleep. There was essentially no change in power spectrum of NREM sleep. These results clearly demonstrated that sleep induced by activation of A2AR in the VLPO is similar to physiological sleep, indicating that VLPO could be used as a potential target for some clinical sleep disorders. antagonist, MSX-3, given at5and10nmol/side into the rat VLPO had no effect on sleep-wake profiles, indicating that A2AR in the VLPO is not involved in the physiological regulation of sleep.Adenosine given at0.5,1.5or4.5nmol/side into the rat VLPO had no effect on sleep-wake profiles. This may have resulted from coincident activation of opposing effects of adenosine at A1R and A2AR, nullifying the general inhibitory effect of adenosine buildup.Fos-immunoreactive (ir) neurons increased in the wake-related areas including locus coeruleus (LC), ventrlateral periaquiductal gray (VLPAG), and tuberomammillary nucleus (TMN) after bilateral microinjection of CPA at1.5nmol/side into rat VLPO. Fos-ir neurons in cortex also increased significantly. These results indicated that promotion of wakefulness induced by activation of A1R in the VLPO is related to the activation of wake-related areas including LC, VLPAG and TMN.Fluorescent immunostaining demonstrated that there is low levels of A1R expression in the VLPO. And the expression of A1R increased after sleep deprivation, which may be responsible for preventing excessive sleepiness.Role of A1R in the VLPO on the regulation of sleep homeostasisSince recovery sleep following sleep deprivation is an essential paradigm to study the homeostatic mechanisms regulating sleep-wakefulness, we examined the effects of bilateral microinjection of CPA into the rat VLPO on recovery sleep after6h of sleep deprivation. Unlike the control group which had an obvious sleep rebound after6h of sleep deprivation, CPA given at1.5nmol/side significantly reduced the total amounts of NREM and REM sleep during recovery for2h after injection, and increased wakefulness. Meanwhile, NREM and REM sleep latency was increased and delta power spectrum of NREM sleep was decreased. Whereas, blockade of A1R in the VLPO with CPT at55pmol/side increased NREM sleep and decreased wakefulness in the second hour after injection. REM sleep was unchanged. These results indicated that A1R in the VLPO plays a crucial role in the regulation of sleep homeostasis.Summary1. A1R in the VLPO is involved in the induction and maintenance of wakefulness under physiological condition.2. Activation of A1R in the VLPO promoted wakefulness, which may be related to the activation of wake-related areas including LC, VLPAG and TMN.3.A1R in the VLPO is involved in the regulation of sleep homeostasis. Activation of A1R in the VLPO may prevent excessive sleepiness after sleep deprivation.4. A2AR in the VLPO is not involved in the physiological regulation of sleep and wakefulness.5. There is low levels of A1R expression in the VLPO. And the expression of A1R increased after sleep deprivation.