Effect And Mechanism Of GABA Of The Posterior Hypothalamus On Sleep-wakefulness Cycle In Rats

Background and Aim:The neurons containing gamma-aminobutyric acid(GABA) play an important role in the alternation from wakefulness to sleep.GABAergic neurons are not only located in the ventrolateral preoptic area(VLPO)but also in the tuberomammillary nucleus(TMn)of the posterior hypothalamus.However, GABAergic neurons in TMn have not received much attention for their role in regulation of sleep-wakefulness cycle.The aim of this study is to investigate the effect of GABAergic neurons in the TMn on sleep-wakefulness cycle.METHODS:Pathogen-free adult male Sprague-Dawley rats(250-300 g)were randomized into 3 groups:control group(n=5),VLPO lesion group(n=5)and VLPO lesion plus bicuculline treated group(n=5).Rats were anesthetized with pentobarbital (35 mg/Kg,ip),the skull were exposed.4 EEG screw electrodes were implanted into the skull,in frontal(two)and parietal bones(two)of each side,and three flexible EMG wire electrodes were placed into the nuchal muscles.The free ends of the leads were soldered into a socket.Guide cannular for microinjection were implanted into the adjacent of TMn(AP-4.2 mm,ML±1.3 mm,DV-7.4 mm).All implantations were affixed to the skull with dental cement,except the area(0.5 cm)around the bregrna filling with bone wax that was removable.One week after surgery,the sockets were connected via flexible recording cables to EEG polygraph(EEG-5208,Nihon Kohon Corporation,Tokyo,Japan),signals were digitized by CED Micro 1401 MKⅡ(Cambridge Electronic Design Limited,London,UK)and computer running the Spike 2(CED)recording system.A 24 h basic wake-sleep states in control group were recorded and analyzed by manually scored in 30 s epochs on the digitized EEG/EMG. On the 8th day,a fine glass micropipette(10-20 mm tip diameter)containing ibotenic acid solution for specific cell damage(10 nM/0.5μl,Sigma)was injected into the VLPO on each side after bone wax removed under anesthetization.One week after the second surgery,rats of VLPO lesion group were recorded sleep-wakefulness states for two weeks.On the 10th day after lesion of VLPO,bicuculline(10 nM/0.5μl),a GABAA-receptor antagonist,was microinjected into the TMn and recorded sleep-wakefulness states for 24 h.RESULTS:(1)The ratio during 24 h of W,SWS1 were respectively increased by 13.17%(P＜0.01),28.9%(P＜0.01)and SWS2,PS were respectively decreased by 43.74%(P＜0.01),44.07%(P＜0.01)in VLPO lesion group on the 9th day compared to before lesion.(2)Microinjection of bicuculline into the TMn of VLPO lesion animals at 10:00 am on the 10th day elicited a wake for 40-55 min with a latency of 15 min. Whereas,24 h sleep-wake states showed that the ratio of W,SWS1 were respectively increased by 12.61%(P＜0.01),50.97%(P＜0.01),SWS2,PS were respectively decreased by 68.08%(P＜0.01),39.92%(P＜0.05)compared to before VLPO lesion.CONCLUSION:The evidence of deep slow wave sleep significant decrease induced by VLPO lesion suggests that GABAergic neurons in the VLPO play an important role in maintaining sleep.The data of bicuculline,a GABA_A-receptor antagonist,microinjection into TMn after VLPO lesion elicited wakefulness and sleep depression for 50 min,by contraries increased significantly light slow wave sleep for 24 h suggest that the GABAergic neurons in the TMn relate to sleep drive(sleepiness) via local circuit to inhibit directly histaminergic neurons.