Endogenous Adenosine Suppresses The Medial Parabrachial Nucleus To Promote Sleep Via Adenosine A₁ Receptors

Aim: Adenosine,widely spread in the brain,was regard as a classical neurotransmitters regulating endogenous sleep.Endogenous adenosine promoted sleep by inhibiting the TMN histaminergic neurons and the LH orexin neurons via adenosine A₁ receptors.In addition,adenosine is an important neurotransmitters in studying sleep homeostasis mechanism.Sleep deprivation enhanced the concentration of adenosine in animal brain,and recovery sleep can reduce the concentration of adenosine.The PB glutamate neurons is crucial to promote awakening,activating which can maintain animal wakefulness.Cell-specific lesions of the PB produced deep coma,The MPB was considered to be the key nuclei to produce physiological arousal.The MPB adenosine A₁ receptors may be sleep homeostasis targets.This experiment,using the method of pharmacology,reveals the function and mechanism of adenosine in the MPB in regulating sleep-wake cycle.Furthermore,whether adenosine in the MPB be involved in sleep homeostasis mechanism.Methods: To investigate the effects of adenosine on physiological sleep-wake behavior,we undertook pharmacological manipulation on MPB neurons.Another group of rats were implanted with guide cannulae bilaterally which were just 1 mm above the MPB location and EEG/EMG recording electrodes.Recording EEG/EMG after one week recovery.?1?adenosine at a dose of 0.5,1.5,4.5 nmol /side was microinjected into MPB at21:00.Rats can be divided into four groups,ACSF was microinjected into the MPB in the control group in 21:00,meanwhile,the other three groups were given adenosine at a dose of 0.5,1.5,4.5 nmol /side.Observing the change of rats in sleep behavior.Finally,confirming cannula position with nissl staining.?2?In order to detect the effect of endogenous adenosine in the MPB on sleep behavior,we did the DAB staining of MPB firstly,and then the experimental rats were divided into two groups,ACSF and 2.0 nmol adenosine deaminase inhibitor EHNA were microinjected into the two groups to observe the effect of endogenous adenosine in sleepawakening.Finally,confirming cannula position with nissl staining.?3?Aimed to explore the mechanism of adenosine in the MPB in sleep-wake regulation.Adenosine A₁ receptor agonist CPA at a dose of 1.5 nmol were microinjected into the MPB,with the control group given with the same volume of ACSF,then regording the change of sleep behavior to explore the sleep effect of adenosine in the MPB.Finally,confirming cannula position with nissl staining.?4?The effect of the MPB adenosine A₁ receptors in the physiological sleep behavior is unknown.In order to block the effect of the physiological adenosine acting on the MPB adenosine A₁ receptors,we microinjected 1.7 nmol adenosine A₁ receptor antagonist CPT into the MPB,with the control group given with the same volume of ACSF.Confirming cannula position with nissl staining.?5?adenosine is the most important neurotransmitter in sleep homeostasis,this study foxs on whether adenosine in the MPB participate in sleep homeostasis.Sleep deprivation for Six hours before microjection?13:00-19:00?by using "light touch" to activate sleep homeostasis in rats.Then,CPT and drugs solvent were given to all of the rats at 19:00 to observe the difference of sleep rebound between the two groups.Identifying whether MPB adenosine A₁ receptors participate in sleep homeostasis.Perfusion and nissl staining after recording.Result:?1?The sleep latency shortened by 98.7%?20.1 ± 5.99 min vs 10.1 ± 4.6 min,P <0.01?after Adenosine at a dose of 4.5nmol been weremicroinjected,comparing with the control group.the amount of wakefulness in 2 h decreased by?48.5 ± 8.4 min vs 92.5 ± 5.6min,P < 0.01?,Concomitantly,the NREM sleep increased by 149.1%?57.8 ± 5.6 min vs23.2 ± 4.6 min,P < 0.01?,but REN sleep did not change?13.7 ± 3.6 min vs 4.4 ± 1.4min,P < 0.001?.Meanwhile,the amount of wakefulness decreased by 22.8%?71.4 ± 5.9min vs 92.5.3 ± 5.6 min,P < 0.05?and the NREM sleep increased by 89.2%?43.9 ± 4.8min vs 23.2 ± 4.6 min,P < 0.05?.But REM sleep did not change.And adenosine at a dose of 0.5 nmol did not impact the sleep-wake regulation in rats.?2?Immune dyeing confirmed the existing of adenosine deaminase in the MPB.The group of adenosine deaminase inhibitor administration decreased wakefulness by 31.0%?31.0 ± 2.1 min vs 44.9 ± 4.1 min,P < 0.05?in the second hour,with 85.2%?46.3 ± 3.5min vs 25.0 ± 7.1 min,P < 0.05?increment in NREM sleep in 2 hours,compared with the control group,and REM sleep did not change.?3?In the experience of CPA microjection,the amount of wakefulness decrease by30.7%?84.9 ± 5.1 min vs 122.5 ± 9.4 min,P < 0.05?with the NREM sleep increased by63.2%?84.0 ± 4.7 min vs 51.5 ± 7.7min,P < 0.05?,weanwhile,REM sleep did not change.?4?In the experience of CPT microjection,the amount of wakefulness enhanced by46.3%?38.2 ± 2.4 min vs 26.1 ± 4.5min,P < 0.05?,with the NREM sleep decreased by35.3%?21.1 ± 2.0 min vs 32.6 ± 4.3min,P < 0.05?in the first hour after CPT microjection.?5?The phenotype with sleep rebound inhibition maintained in 2 hours after CPT administration with 43.8%?25.0 ± 6.0 min vs 44.5 ± 5.8 min,P < 0.05?decrement in NREM,compared with the control group.and REM sleep decreased,too.Conclusions: Adenosine in the MPB shorten the sleep latency and induced NREM sleep.Adenosine of the MPB induce sleep by exciting adenosine A₁ receptor.In comparison,blocking the adenosine A₁ receptors in the MPB induced wakefulness.adenosine A₁ receptors in the MPB participant regulating sleep homeostasis,and blocking the adenosine A₁ receptors of the MPB can inhibit sleep rebound after sleep deprivation.