Study On The Sleep-wake Cycle Affected By Adenosine In The Ventrolateral Preoptic Area Of Rats

ObjectiveAdenosine (AD) is an important hormonal factor which takes part in sleep-wakefulnessregulation. The hypothalamic ventrolateral preoptic area (VLPO) has an importantregulatory effect on sleep. In this study we examined the effect of AD onsleep-wakefulness regulation in the VLPO of rats.Methods1. Stereotaxic surgery Screws were implanted into the parietal and occipital bonesfor electroencephalogram (EEG) recording and two silver electrodes were placed underthe nuchal muscles for electromyogram (EMG) recording. According to the atlas ofPaxinos and Watson, two guide cannulae were stereotaxically implanted into theVLPO for drug microinjection. All electrodes and guide cannulae were secured withdental cement.2. EEG/EMG recording and scoring The EEG/EMG signals were recorded byMP150data acquisition system and assorted into wake, non-rapid eye movement(NREM) sleep, and rapid eye movement (REM) sleep by Sleepsign software in10-secepochs to analysis time-course changes of sleep-wakefulness cycle; total time spent onwake, NREM and REM sleep, and total sleep time (TST); changes in number of wake,NREM and REM sleep bouts at different ranges of episode duration; stage transitionnumbers from NREM sleep to REM sleep, REM sleep to wakefulness, and numbersbetween NREM sleep and wakefulness.3. Pharmacological treatment Drugs (experimental groups) or ACSF (control group) were administrated into the VLPO at10:00a.m.(2h after lights on) through the guidecannula which connecting with an injection pump in one miniute. The volume ofmicroinjection was0.5l. The needle was kept in the guide cannula about1min toprevent the physic liquor flow out. Fake injects were started two days before theexperiment. When multiple drugs were applied in the same day, the interval betweeninjections was10-20miniutes.4. Morphological methods (1) The brains were removed and embedded intoparaffin, and the immunohistochemical detection was used to detect A1and A2Areceptors in the VLPO.(2) Brain coronal sections were cut on a freezing microtome,and stained with thionine to visualize the cannula tracks and injection sites.5. Total sleep deprivation The rats were gently touched, when the rats wereimmobile for3-4s without any gross body, head or whisker movements and its EEGshowed a synchronization activity.6. Statistical analysis All statistical analysis was performed using SPSS10.01software. The significance of differences was analyzed by one-way ANOVA followedby the LSD test (comparisons between control and experimental groups) or the S-N-Ktest (multiple comparisons between groups) as posthoc analyses. Independent Student’st-tests were used for comparison between two groups. Data were expressed as mean±SEM. Differences with P <0.05were considered statistically significant.Results1. Effect of increasing AD levels on sleep-wakefulness in the VLPO1.1Increased arousal was observed following AD administration at doses of3.0and4.5nmol/side, which began at2h post-injection and lasted for4h. During the5h afterinjection, AD at3.0or4.5nmol/side increased wakefulness time, reduced NREM andREM sleep time, and TST; AD (at3.0or4.5nmol/side) increased the number of longwake bouts and short NREM sleep bouts, but decreased the number of medium REM and NREM sleep bouts. AD at4.5nmol/side also decreased the number of short REMsleep bouts, and long NREM sleep bouts; AD (at3.0or4.5nmol/side) decreased the5hstage transition numbers from NREM sleep to REM sleep and REM sleep towakefulness, but did not affect the stage transition numbers between NREM sleep andwakefulness. AD at1.5nmol/side had no significant effect on the sleep-wake cycle.These results indicated that increased exogenous AD levels in the VLPO produced anincrease in wakefulness and a decrease in sleep.1.2When given at0.1nmol/side, NBTI (an AD transport inhibitor) increased thewakefulness starting at2h after injection and lasted for3h. During the5h afterinjection, NBTI increased wakefulness time, reduced NREM and REM sleep time, andTST; NBTI increased the number of long wake bouts, decreased the number of mediumREM and NREM sleep bouts; NBTI did not affect the5h stage transition numbers fromNREM sleep to REM sleep, REM sleep to wakefulness, and numbers between NREMsleep and wakefulness. The results suggested that increased endogenous AD levels inthe VLPO also increased wakefulness and suppressed sleep.2. The expression of adenosine receptors in the VLPO of rats. The brains wereremoved and embedded into paraffin, immunostaining for detection of A1and A2Areceptors in the VLPO with A1receptor and A2Areceptor antibody. The expression ofA1receptors could be detected in the VLPO, but A2Areceptors seemed to beundetected.3. Effects of A1R on sleep-wakefulness in the VLPO3.1When given at0.29,0.72, and1.43nmol/side, CHA (an A1receptor agonist)increased wakefulness starting at2h post-injection and effects at each of the dosesabove lasted for1,3, and4h, respectively. During the5h after injection, CHA given at0.72or1.43nmol/side increased wakefulness time, reduced NREM and REM sleeptime, and TST; CHA (0.72or1.43nmol/side) caused an increase in the number of longwake bouts, a reduction in the number of short wake bouts, and medium REM and NREM sleep bouts. CHA given at1.43nmol/side also decreased the number of longNREM sleep bouts; CHA (0.72or1.43nmol/side) decreased the5h stage transitionnumbers from NREM sleep to REM sleep and REM sleep to wakefulness, but did notaffect the stage transition numbers between NREM sleep and wakefulness. Theseresults demonstrated that the selective activation of A1R in the VLPO increasedwakefulness and reduced sleep.3.2When given at0.565nmol/side, CPT (an A1receptor antagonist) increasedNREM sleep starting at3h post-injection and effects lasted for3h. During the5h afterinjection, CPT decreased wakefulness time, while increased NREM sleep and TST; CPTdecreased the number of medium wake bouts, and the number of short and mediumNREM sleep bouts, whereas increased long NREM sleep bouts; CPT decreased the5hstage transition numbers between NREM sleep and wakefulness, but did not affect thenumbers from NREM sleep to REM sleep and REM sleep to wakefulness. These resultsindicated that administration of A1receptor antagonist into the VLPO produced areduction in wakefulness and an increase in sleep.3.3Compared with the ACSF+AD group, coinjection of CPT (0.565nmol/side) andAD (4.5nmol/side) into the VLPO decreased the time of wakefulness starting at2hafter injection with effects lasting for4h. During the5h after injection, compared withthe ACSF+AD group, coinjection of CPT and AD decreased wakefulness time, butincreased NREM and REM sleep time, and TST; Compared with the ACSF+AD group,coinjection of CPT and AD decreased the number of long wake bouts, increased thenumber of medium REM sleep bouts, and increased the number of medium and longNREM sleep bouts; Coinjection of CPT and AD increased the5h stage transitionnumbers from NREM sleep to REM sleep and REM sleep to wakefulness, but did notaffect the numbers between NREM sleep and wakefulness. These results demonstratedthat blocking A1receptor effectively diminished AD-induced wakefulness.4. The possible intracellular signal transduction pathways of AD receptors 4.1During the5h after injection, cAMP (2g/l) decreased wakefulness time,increased NREM sleep time and TST. However, cAMP did not affect REM sleep time.4.2During the5h after coinjection, compared with the ACSF+AD group,coinjection of U-73122(5mol/L) and AD (4.5nmol/side) decreased wakefulness time,enhanced REM sleep time and TST. However, coinjection of U-73122and AD did notaffect NREM sleep time.5. The role of adenosine on sleep homeostasis regulation in the VLPO5.1After the rats were total sleep deprived for6h, compared with the SD+ACSFgroup, SD+NBTI (0.1nmol/side) group enhanced wakefulness and decreased NREMsleep in the first hour, and decreased REM sleep in the second hour. During the2h aftercoinjection, SD+NBTI group increased wakefulness time, reduced NREM and REMsleep time, and TST; SD+NBTI group increased the number of long wake bouts,whereas decreased the number of medium REM and NREM sleep bouts; The5h stagetransition numbers from NREM sleep to REM sleep, REM sleep to wakefulness, andnumbers between NREM sleep and wakefulness were not affected. These resultssuggested that AD participated in the regulation of sleep homeostasis in the VLPO.5.2After the rats were total sleep deprived for6h, compared with the SD+ACSFgroup, SD+CPT (0.565nmol/side) group decreased wakefulness and increased NREMsleep in the first hour. During the2h after coinjection, SD+CPT group decreasedwakefulness time and increased NREM sleep time and TST; SD+CPT group increasedthe number of long NREM sleep bouts; The5h stage transition numbers from NREMsleep to REM sleep, REM sleep to wakefulness, and numbers between NREM sleep andwakefulness were not affected. These results suggested that A1R involved in theAD-mediated regulation of sleep homeostasis in the VLPO.Conclution1. AD participates in the sleep-wakefulness regulation in the VLPO. 2. AD promotes wakefulness via A1R in the VLPO.3. Receptor-G protein-AC pathway and receptor-G protein-PLC pathway probablymediate the wakefulness-promoting effect of A1R.4. AD and its A1R in the VLPO involve in the regulation of sleep homeostasis.