Effects Of Microinjecting GABA_A-receptor Agonist Into The Tuberomamillary Nucleus On The Sleep-wakefulness Cycle In The Rat

Background:In the brain,GABAergic neurons were location in the basal forebrain (BF),the preoptic area(POA) and the tubermammillary nucleus(TMN) among those regions.As kown to the GABAergic neurons play an important role in promting and maintain sleep.It is well known the histaminergic(HAergic) neurons are exclusively locationed in the TMN and play an important role in promting and maintain sleep,the neurons contains GABA_A receptor,the ventrolateral preoptic area(VLPO), approximately 80%of neurons in the population contain the inhibitory neurotransmitters GABA,are responsible in the sleep.In my past study,the GABAergic neurons in the TMN play arole in sleep drive,moreover microinjecting GABA_A receptor agonists into the TMN,sleep-wakefulness cycle and electroencephalogram(EEG) feature remains unclear.Aim:The present study is to investigate the effects of muscimol,a potent agonist of GABA_A-receptor,on the sleep-wakefulness cycle and on histamine-induced arousal acting in the TMN in the freely rats,and anaylze the change of the power spectrum.The aim to furthered study that the interaction between TMN HAergic and GABAergic neurons for modulating sleep-wakefulness cycle and that the EEG feature in the process.Methods:Sprague-Dawley rats were randomly divided into control group(n=7), saline group(n=7),muscimol 0.5μg group(n=6),muscimol 0.25μg group(n=4),HA group(n=7) and HA plus muscimol group(n=5).Four electrodes(2mm in diameter) were implanted into the skull to the surface of the cerebral dura mater for recording EEG and three electrodes were inserted bilaterally into the dorsal neck muscles for recording electromyogram(EMG).The stainless steel guide cannular(0.6mm in diameter) was implanted into the adjacent of TMN(anteroposterior, -4.3 mm;mediolateral,±1.3 mm;and dorsoventral,-7.4mm with reference to the bregma,2mm above the TMN) for subsequent drug microinjection.After one week recovery,the sockets were connected via flexible recording cabels to EEG polygraph,signals were digitized by Micro 1401 MKâ…¡(Cambridge Electronic Design Limited(CED),London,UK) and computer running the spike 2(CED) recording system.Muscimol was microinjected into bilateral TMN from 22:00 and to 22:20 and PSG was recorded at 20:00.HA was microinjected from 10:00 to 10:20 and PSG was recorded at 8:00.When multiple drugs were applied in the same day,the first drug was given from 10:00 to 10:20 and the second one was given from 12:00 to 12:20 and PSG was recorded at 8:00.The wake and sleep states were statistically calculated per 30 sec for 24 h.The power spectral density(PSD) was averaged over 30-sec epochs for the frequency range of 1-60 Hz using a fast fourrier transform routing and the Spike 2,and correlated with the parameters of the sleep/wake stages.In preliminary power spectrum analyses,frequency band ranges were set for the duration at the following ranges:δ:1-4 Hz,θ:4.2-8 Hz,α:8.2-12 Hz,β:12.2-30 Hz(β₁:12.2-20 Hz,β₂: 20.2-30 Hz) andγ:30.2-60 Hz.Results:1.Changs of sleep/wake stages:(1) As compare with the saline group, muscimol 0.25μg produced a marked increased light slow wave sleep(SWS1),deep slow wave sleep(SWS2)(22.09%and 62.08%),while wakefulness(W) decreased (10.98%),and muscimol 0.5μg produced that SWS1,SWS2 increased(29.34%and 114.43%) and W decreased(19.46%).Paradoxical(PS) had no effect significantly as compared to the saline.The dose-dependency of slow wave sleep(SWS) promotion by the muscimol administration is summarized.In addition,muscimol cause hypersomnia and a marked increase ofδband,but suppressβband.(2) HA into the TMN,elicited a wake for above 3h with a latency of 37min, meanwhile 12h sleep-wake states showed that W were increased,SWS and PS were decreased compare to saline group.Microinjection of muscimol 0.5μg into the TMN after HA injection,increase SWS and PS and also reversed the HA induced wakefulness to sleep.In addition,HA cause behavioral excitation and EEG activation with a long lastingβ₂ band and with suppression ofα,θandδband.Therefore,muscimol into the TMN suppresses the effects of HA by restoringδband and inducing SWS,these effects being accompanied by all behavioral signs of sleep.2.Change of bout:The muscimol treatments decreased the number of the shorter S1 bouts(＜120 sec) and increased the number of the prolonged one(120-240 sec and＞240 sec) in a dose-depend manner and increased the number of the prolonged S2(120-240 sec and＞240 sec) and decrease the number of the prolonged wakefulness(＞240 sec) compare to the saline injection.On the other hand,the muscimol administration did not affect the numbers of stage transition from W to SWS,from SWS to W,from SWS to PS,from PS to W.Conclusions:1.Muscimol into TMN induce an increasement of SWS by increasingδband,and inhibitingβband,the effect suggest that GABAergic neurons in the TMN play a role in inhibit W and increase SWS,but PS have no effect.2.Muscimol,a potent agonist of GABA_A-receptor,can induce sleep through extension the duration of the corresponding states,rather than increasing the number of conversion in the TMN.3.HA in the TMN play a key role of promoting and maintaining wakefulness by increasingβ₂ band,and decreaed the SWS and PS by suppression ofα,θandδband.4.Muscimol can reverse HA-induced wakefulness into sleep,and that theβ₂ band by HA-induced had been inhibited andδband increased is the character.The GABAergic neurons and HAergic neurons of the TMN may have local loop,involved in the regulation of sleep and arousal.