Objective Neurons containing gamma-aminobutyric acid (GABA) play an important role in the regulation from wakefulness to sleep.Recent evidences have demonstrated that the interaction between GABAergic neurons in ventrolateral preoptic area (VLPO) and histaminergic (HA) neurons in the tuberomammillary nucleus (TMN) play a key role for regulating sleep-wakefulness cycle.GABAergic neurons also in the TMN have been recently documented.However, GABAergic neurons in TMN have not received much attention for their role in sleep-wakefulness cycle.The present study was to investigate the effect of inhibition of GABA uptake in the TMN nucleus on sleep-waking states following the VLPO lesion and further understand the role of the GABAergic neurons in the TMN in sleep-wakefulness cycle.Methods Thirty adult and male SD rats were used in this study.Under anesthesia, 4 EEG screw electrodes were implanted into the skull and 3 flexible EMG wire electrodes were placed into the nuchal muscles.After 24 h basal record of sleep-wake cycle, microliter syringes containing ibotenic acid (IA, Sigma, St.Louis, USA) solution (10 nmol/0.5 μL/side) were bilaterally injected into the VLPO for specific cells lesion.After VLPO lesion, nipecotic acid (NPA, Sigma, St.Louis, USA), a GABA uptake inhibitor (1 mmol/0.5 μL/side), was bilaterally injected into the TMN at 10:00 or 22:00 h for observing the changes of sleep-waking states in light period (8:00-20:00 h) and dark period (20:00-8:00 h) on day 8, 10, 12 and 14, respectively.After recording, histological Nissl staining and immunohistochemical Fos and glutamate decarboxylase (GAD) staining were respectively performed for determining the loss number of cell and GABA neurons in the VLPO.GAD immunoreactivity (IR) and histidine decarboxylase (HDC)-IR were performed determining the distributions and morphologic characteristics of GABAergic and HAergic neurons in the TMN.Results (1) Effect of VLPO lesion on sleep-waking cycle and cortical EEG power.After VLPO lesion, the sleep-waking cycle was consistently recorded for 22 days, we found that the average time per day in wake (W) from day 6 to 16 and light slow wave sleep (SWS1) from day 8 to 22 were significantly increased, however, deep slow wave sleep (SWS2) from day 4 to 22 was significantly decreased, paradoxical sleep (PS) time did not change compared with pre-lesion, respectively.During recording days, the increased W and decreased SWS2 time was only observed in dark period from day 8 to 14.The VLPO lesion induced an maximal variation in sleep-waking cycle on day 12, the W and SWS1 time were respectively increased by 39.59% (P<0.01) and 46.86% (P<0.01), SWS2 was decreased by 50.14% (P<0.01) in light period, W time was increased by 15.95% (P<0.05) and SWS2 was decreased by 57.5% (P<0.01) in the dark period compared with pre-lesion.The numbers of Fos-IR and GAD-IR neurons were decreased by 75%-90% following IA bilateral microinjection into the VLPO in this study.The cell loss in the VLPO was linearly associated to SWS2 time.In addition, VLPO lesion also induced a significant increase in EEG β and α power, and a significant decrease in EEG δδ power in light period compared with pre-lesion.(2) Effect of inhibition of GABA uptake in the TMN on sleep-waking states and cortical EEG power following the VLPO lesion.Double immunostaining revealed GAD-IR and HDC-IR neurons almost coexistence in the TMN.NPA bilateral mieroinjected into the TMN on day 8, 10, 12, and 14 after VLPO lesion at 10:00 h induced a decrease in SWS1 by 23.53% (P<0.05) and an increase in SWS2 by 83.41% (P<0.01) in light period, and at 22:00 h induced a decrease in W by 20.22% (P<0.05), an increase in SWS1 by 38.38% (P<0.05) and SWS2 by 113.1% (P<0.01) in dark period compared with VLPO lesion treated with vehicle; an increase in W by 25.61% (P<0.05) and SWS1 25.32% (P<0.05) and a decrease in SWS2 by 25.87% (P<0.05) in light period, a decrease in W by 8.90% (P<0.05) and an increase in SWS1 43.52% (P<0.05) in dark period, compared with VLPO pre-lesion, respectively.Furthermore, NPA microinjection into the TMN following the VLPO lesion induced a significant increase in cortical high-voltage electrical activity with dominant waves in the δ and α band power, and a decrease in β power, and a sleep for 5 h with a latency of 45 minutes.Conclusion The neurons containing GABA in hypothalamus are not only in the VLPO, but also in the TMN.In celluar morphology, GABA coexists with HAergic neurons in the TMN.The GABAergic neurons in the VLPO play a key role in maintaining slow wave sleep as evidence by the decreased SWS2 following the VLPO lesion.The loss in numbers of GABA neurons in the VLPO was linearly associated to the reduction of SWS2 time.The GABAergic neurons in the TMN is related to sleep drive (sleepiness) as evidence by the increased SWS 1 after the VLPO lesion and by increased SWS2 response to local inhibition of GABA uptake following the VLPO lesion, even though the increase of SWS2-induced by inhibition of GABA uptake does not completely alter the phenomenon of SWS1 increase-induced by VLPO lesion.