The Projection Between Ventrolateral Preoptic Area And Tuberomammillary Nucleus On The Regulation Of EEG Activity Of Rats

ObjectiveVentrolateral preoptic area (VLPO) and tuberomammillary nucleus (TMN) are criticalregions involving in sleep-wakefulness regulation in the central nervous system.γ-aminobutyric acid (GABA) and Triprolidine（Trip）are key neuro-hormonal factorswhich modulate sleep-wakefulness cycle. Brain stereotaxic, nucleus spile,microinjection and polysomnography technologies were used to observe and record thevariations of EEG activity in rat sleep-wakefulness cycle, and also investigate theeffects of excitatory neurotransmitter L-glutamate (L-Glu) and GABAAreceptorantagonist Bicuculline (Bic), H1receptor antagonist Triprolidine（Trip）in this study.Methods1. Animal model Adult male SD rats (SPF grade) weighing270-290g were used.After anesthetized by pentobarbital (50mg·kg–1, i.p.), electroencephalogram (EEG)and electromyography (EMG) electrodes were implanted for polysomnographicrecording, and two stainless steel guide cannulas were inserted into VLPO (AP:-0.36mm; R:1.30mm; H:-7.00mm) and TMN (AP:-3.96mm; R:1.50mm; H:-7.70mm) for drug application in rats. Guide cannulas and recording electrodeswere fixed to the skull surface with dental cement. Each animal needed7days forrecovery in a soundproof recording room after surgery operated, then wasconnected to an EEG/EMG recording cable and habituated for3days beforepolysomnographic recording. 2. Grouping Rats were randomly divided into seven groups: night control group(Both TMN and VLPO are microinjected with ACSF, TMN+ACSF&VLPO+ACSFgroup, n=8) and night experimental groups [(1) microinjection of ACSF andL-Glu into TMN and VLPO respectively (TMN+ACSF&VLPO+L-Glu group, n=7);(2) microinjection of Bic and ACSF into the TMN and VLPO respectively(TMN+Bic&VLPO+ACSF group, n=7);(3) microinjection of Bic and L-Glu intoTMN and VLPO respectively (TMN+Bic&VLPO+L-Glu group, n=8)]；daycontrol group (Both VLPO and TMN are microinjected with ACSF,VLPO+ACSF&TMN+ACSF group, n=7) and day experimental groups [(1)microinjection of ACSF and L-Glu into VLPO and TMN respectively(VLPO+ACSF&TMN+L-Glu group, n=8);(2) microinjection of Trip and ACSFinto the VLPO and TMN respectively (VLPO+Trip&TMN+L-Glu group, n=7)].3. Microinjection ACSF or drugs (L-Glu, Bic, Trip) of1μl was microinjected intoTMN and VLPO through a stainless steel guide cannula at injection rate of1μl·min-1, and the needle was kept in the cannula for1min to prevent the physicliquor overflow. Drug application was performed at22:00-22:20(10:00-10:20).4. Polysomnography Polysomnography (PSG, including EEG and EMG) wasstarted2hours before drugs application at20:00or08:00and sustained24hours.According to the PSG results, each10s were regard as one epoch. Thesleep-wakefulness cycle is divided into three stages:(1) Wakefulness (W);(2)Non-rapid eye movement (NREM) sleep;(3) Rapid eye movement (REM) sleep.Total sleep time (TST) was composed of NREM and REM.5. Histological identification Brain coronal sections were cut on a freezingmicrotome, and the cannula track and injection sites were verified with Nissl staining. Rats with inappropriate placements were excluded from the analyses.6. Statistical analyses SPSS17.0software was used for statistical analyses. Theexperimental data were presented as mean±SEM (x±se). Multiple comparisonsbetween groups were analyzed using one-way ANOVA and those at P <0.05wereconsidered as the level of significance.Results1. Compared with the TMN+ACSF&VLPO+ACSF group (n=8), the amount ofwakefulness was decline21.6%(163.71±8.17vs208.83±7.89, P <0.01) alongwith the enhancement of NREM sleep56.4%(112.61±7.37vs71.99±6.34, P <0.01) and TST49.5%(136.29±8.16vs91.17±7.87, P <0.01) after L-Glumicroinjected into VLPO (n=7) in5hours, while there is no significant change inREM sleep.2. Compared with the TMN+ACSF&VLPO+ACSF group (n=8), microinjection ofBic into TMN in TMN+Bic&VLPO+ACSF group (n=7) causes rats’ wakefulness,however REM sleep and NREM sleep had no significant changes.3. Compared with the TMN+Bic&VLPO+ACSF group (n=7), there is no significantchanges of rat sleep-wakefulness cycle in TMN+Bic&VLPO+L-Glu group (n=8).Compared to those of L-Glu microinjected into VLPO alone in theTMN+ACSF&VLPO+L-Glu group (n=7), the wakefulness was increase25.6%(163.71±8.17vs205.56±10.12, P <0.01), meanwhile, REM sleep52.1%(23.67±2.78vs11.33±2.78, P <0.01), NREM sleep26.2%(112.61±7.37vs85.09±8.09, P <0.05) and TST30.7%(136.29±8.16vs94.42±10.12, P <0.01) weredecrease after L-Glu microinjected into VLPO followed by the Bic microinjected into TMN (n=8) in5hours.4. Compared with the VLPO+ACSF&TMN+ACSF group (n=7), the amount ofwakefulness was increase58.0%(113.35±4.32vs179.00±13.91, P <0.01) alongwith the reduction of NREM sleep33.4%(172.05±3.57vs114.51±13.07, P <0.01) and total sleep time (TST)35.2%(186.63±4.32vs120.99±13.91, P <0.01)after L-Glu microinjected into TMN (n=8) in5hours, while there is no significantchange in REM sleep.5. Compared to those of L-Glu microinjected into TMN alone in theVLPO+ACSF&TMN+L-Glu group (n=8), the wakefulness was decrease30.2%(179.00±13.91vs125.03±7.68, P <0.01), meanwhile, REM sleep199.3%(6.48±2.68vs19.40±2.85, P <0.01), NREM sleep35.9%(114.51±13.07vs155.57±6.80, P <0.05) and TST44.6%(120.99±13.91vs174.97±7.66, P <0.01) wereincrease after L-Glu microinjected into TMN followed by the Trip microinjectedinto VLPO (n=7) in5hours.ConclusionVLPO could inhibit the TMN neuronal activity via GABAAreceptor and induce asleep-promoting effect; the enhancement of functional activity of neurons within TMNplays a key role in the generation and maintenance of wakefulness.